The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

El siguiente protocolo se ajusta a las directrices del Consejo Canadiense de Cuidado Animal y ha sido aprobado por la Facultad de Medicina y del Comité de Cuidado de Animales de Farmacia de la Universidad de Toronto. 1. Preparar Obligatorio reactivos como se indica en la Tabla 1 <ol><li> Añadir proteasa y fosfatasa (si es necesario) inhibidores a todas las soluciones de sacarosa, tampones y ddH2O a concentraciones descritas en la Tabla 1. IMPORTANTE: Realice todos los pasos a 4 ° C y pre-cool todos los reactivos y equipos antes del inicio del experimento. Etiquetar todos los tubos, incluyendo tubos de ultracentrífuga, con marcador permanente. </li></ol> 2. Disección de la región cerebral adecuada <ol><li> Sacrificar el animal por dislocación cervical o decapitación. Casa y eutanasia de animales de acuerdo con las políticas institucionales y gubernamentales en el cuidado de animales. </li><li> Eliminar rápidamente ªe cerebro del cráneo y el lugar en un bloque de disección fría. </li><li> Diseccionar la región cerebral correspondiente de tejido fresco y vaya al paso 3. </li></ol> 3. homogeneización de tejido con accionado por motor de vidrio-teflón Homogeneizador <ol><li> Coloque 50-100 mg (por SPM), o 100-200 mg (por PSD) del tejido diseccionado en un tubo de polipropileno de 13 ml marcado que contiene 4 ml de solución de sacarosa 0,32 tamponada con HEPES M. Transferir la muestra a un 15 ml cónico tejido de vidrio-teflón grinder / homogeneizador, establezca la unidad de motor a 900 rpm (posición 7) y homogeneizar la muestra con 12 golpes en un período de 30 segundos. Utilice un homogeneizador de vidrio-teflón diferente entre las muestras, o enjuagar el homogeneizador con agua destilada fría entre las muestras y seque con un Kimwipe. NOTA: Hasta 4 g de tejido se puede homogeneizar en 4 ml de solución de sacarosa 0,32 tamponada con HEPES M, sin embargo, es aconsejable usar menos tejido para asegurar fraccionamiento adecuado. </li><li> Transferir la muestra homogeneizada espalda to el mismo tubo de polipropileno de 13 ml. Reserva 100 l de homogeneizado y almacenar a -80 ° C para la cuantificación de proteínas y posterior análisis de Western blot de la fracción de proteína total. </li></ol> 4. baja velocidad de centrifugación para eliminar la fracción nuclear (rendimientos sobrenadante S1) <ol><li> Centrifugar el homogeneizado en un rotor de ángulo fijo a 900 xg durante 10 min a 4 ° C. Transferir el sobrenadante (S1) a una nueva, etiquetado tubo de 13 ml y resuspender el precipitado fracción nuclear (P1) en 500 l de sacarosa 0,32 M tamponada con HEPES. NOTA: El (P1) fracción puede ser almacenado a -80 ° C y se utiliza para el análisis de transferencia de Western de la fracción nuclear. </li></ol> 5. enriquecimiento de la fracción bruta sinaptosomal (rendimientos Pellet P2) <ol><li> Centrifugar el sobrenadante (S1) a 10.000 xg durante 15 min a 4 ° C. Eliminar el sobrenadante (S2) y de reserva 500 l en un tubo de microcentrífuga para almacenar a -80 ° C para su posterior quant proteínaficación y análisis de Western blot de la fracción de membrana citosólica / luz. Resuspender el restante sinaptosomal crudo fracción de sedimento (P2) en 1 ml de solución de sacarosa tamponada con HEPES 0,32 M y luego agregar otra solución de sacarosa tamponada con HEPES 3 ml de 0,32 M. </li><li> Se centrifuga a 10.000 x g durante 15 min a 4 ° C. Eliminar el sobrenadante (S2 &#39;) y de reserva 500 l en un tubo de microcentrífuga para almacenar a -80 ° C para la cuantificación de proteínas y posterior análisis de Western blot de la fracción de membrana citosólica / luz. Guarde el lavado de pellets sinaptosomal crudo (P2 &#39;) en el tubo de polipropileno. </li></ol> 6. lisis (Hipoosmótico Choque) de la Fracción crudo sinaptosomal <ol><li> Lyse el pellet sinaptosomal crudo (P2 &#39;) en el tubo de polipropileno mediante resuspensión en 1 ml de ddH 2 O. Añadir otro 3 ml de ddH2O, transfiera a un homogeneizador de tejidos de vidrio-teflón, y homogeneizar a mano con 3 golpes. NOTA: Es importantepara llevar a cabo este paso lo más rápidamente posible y rápidamente vaya al paso 6.2. </li><li> Muestras de traslado desde el homogeneizador de nuevo en el mismo tubo de polipropileno de 13 ml y ajustar rápidamente la muestra de nuevo a 4 mM HEPES con 16 l de una solución 1 M HEPES, invierten para mezclar. </li><li> Girar las muestras a 4 ° C durante 30 minutos para asegurar la lisis completa. </li></ol> 7. Enriquecimiento de la Fracción sinaptosomal Membrana (P3) <ol><li> Centrifugar a 25.000 x g durante 20 min a 4 ° C. Retire el crudo vesicular fracción sobrenadante (S3) y reservar en un tubo de 5 ml para almacenar a -80 ° C para la posterior cuantificación de proteínas y análisis de transferencia Western. Resuspender la fracción de membrana de sinaptosomas (P3) en 1 ml de solución de sacarosa 0,32 tamponada con HEPES M. </li></ol> 8 Preparación del gradiente de sacarosa discontinuo <ol><li> Pipetear exactamente 3,5 ml de solución de sacarosa tamponada con HEPES 1,2 M y exactamente 3,0 ml de cada uno de 1,0 M y 0,8 M HEPESolución de sacarosa S-guardado en separadas 6 ml complemento de tubos con cierre. NOTA: Esto se hace para medir la validez de los volúmenes de las memorias intermedias que se utilizan para hacer que el gradiente de sacarosa. La medición exacta de los volúmenes es importante asegurarse de que los gradientes resultantes serán equilibradas para ultracentrifugación. </li><li> Utilizando una pipeta Pasteur de vidrio y el bulbo, transferir solución de sacarosa 1,2 M tamponada con HEPES a los 12 ml tubo ultracentrifugadora polialómero. Capa cuidadosamente la solución de sacarosa tamponada con HEPES 1,0 M en la parte superior de la solución de sacarosa tamponada con HEPES 1,2 M. Finalmente, la capa de la solución de sacarosa tamponada con HEPES 0,8 M en la parte superior de la solución de sacarosa tamponada con HEPES 1,0 M. Utilice una pipeta Pasteur nueva para cada solución de sacarosa y tener cuidado de no alterar el gradiente de sacarosa. Las vibraciones de un vórtice banco o microcentrífuga esperar perturbaciones de la integridad de la gradiente. </li><li> Utilizando una pipeta Pasteur, la capa de la fracción de membrana sinaptosomal se resuspendió (P3) en la parte superior del gradiente discontinuo de sacarosa preparada.Asegúrese de que los gradientes se equilibran con un peso en el interior del cubo de rotor basculante. Si los cubos no están equilibradas, utilizar una pipeta P200 para agregar cuidadosamente sacarosa 0,32 M tamponada con HEPES a la parte superior del gradiente y el equilibrio de los cubos del rotor. </li></ol> 9. El fraccionamiento de la membrana plasmática sináptica (SPM) <ol><li> Ultracentrífuga en un rotor basculante a 150.000 xg durante 2 horas a 4 ° C. Retire con cuidado los gradientes de sacarosa de los cubos de ultracentrífuga. Uso de un 18 G aguja y una jeringa de 1 ml, perforar el tubo en la parte inferior de la solución de sacarosa tamponada con HEPES interfase 1,0 M / 1,2 M y retirar la capa de membrana plasmática sináptica (SPM). </li><li> Anote el volumen que cada muestra de SPM ocupa en la jeringa de 1 ml. Coloque la capa de SPM recogido en 3,5 ml tubos de ultracentrífuga de pared gruesa, añadir exactamente 2,5 volúmenes de 4 mM HEPES para ajustar la concentración de sacarosa de 1,2 M a 0,32 M. Una vez cada muestra ha sido ajustared a 0,32 M de sacarosa, equilibrar los tubos con 0,32 solución de sacarosa tamponada con HEPES M. </li><li> Ultracentrífuga en un rotor de ángulo fijo a 200.000 xg durante 30 min a 4 ° C. Retire y deseche el sobrenadante. Resuspender el pellet sináptica membrana plasmática (SPM) en 300 l de 50 mM EDTA solución HEPES / 2 mM. Guardar las muestras a -80 ° C hasta su utilización para el Western Blot. </li></ol> 10 Preparación de la Fracción de densidad postsináptica (PSD) <ol><li> Descongele las muestras de SPM en hielo. </li><li> Hacer una solución de 0,54% de Triton X-100 en 50 mM de solución EDTA HEPES / 2 mM. </li><li> En un tubo de poliestireno de 5 ml, se combinan 2,7 ml de Triton X-100 / / solución de EDTA y 300 l fracción SPM HEPES y rotar la muestra durante 15 min a 4 ° C </li><li> Muestras de traslado hasta 3,5 ml tubos de ultracentrífuga de pared gruesa y Centrifugar las muestras a 32.000 xg durante 20 min a 4 ° C. </li><li> Reservar el sobrenadante (Triton X-100 fracción soluble [TS]) y se almacena a -80; ° C. NOTA: En esta etapa, el sedimento representa el &quot;PSD-1T&quot; fracción. Un segundo tratamiento con detergente producirá una &quot;PSD-2T&quot; fracción. Si se desea una fracción PSD-1T, continúe en el paso 10.6. <ol><li> Para producir una fracción PSD-2T, resuspender el sedimento en 3 ml de 0,5% de Triton X-100 en 50 mM HEPES / EDTA 2 mM solución. Gire las muestras durante 15 minutos a 4 ° C. Centrifugar las muestras a 32.000 xg durante 20 min a 4 ° C y continuar con el paso 10.6. </li></ol></li><li> Descartar el sobrenadante y resuspender el postsináptica (PSD) pellet en solución de 50 mM de HEPES / EDTA 2 mM. NOTA: El volumen de resuspensión puede depender del tamaño de la pastilla; Se recomienda un volumen de 50-75 l. Debido a que el PSD fracción es una fracción insoluble en detergente, a menudo es necesario añadir 1-2 l de 0,5% de SDS para permitir la resuspensión completa de proteínas. Guardar las muestras a -80 ° C hasta su utilización para el Western Blot. </li></ol>

<ol>
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E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

The authors have no conflicts of interest to disclose.

Hay varios pasos en el proceso que son críticos para un resultado exitoso. En el paso 3, es importante que un grado consistente de homogeneización se logra para cada muestra. Cuando la homogeneización de tejido con el homogeneizador accionado por motor, una velocidad constante se utiliza no sólo para la rotación de la mano del mortero, pero también con el número de golpes. El tiempo de incubación durante el choque osmótico debe ser preciso, ya que la homogeneización ampliada o de la incubación en la solución...

Las neuronas se comunican a través de las sinapsis, y la calidad de esta comunicación está regulada en gran medida por las alteraciones en la composición de las proteínas en la sinapsis. En particular, las proteínas localizadas en la densidad post-sináptica participan en la comunicación neuronal por receptores de neurotransmisores íntimamente andamiaje con sus sistemas de transducción de señales 1. Además, los cambios duraderos en la fuerza de la eficacia sináptica son controlados por la adición o eliminación de los receptores a la densidad post-sináptica 1-6. Por lo tanto, el aislamiento y la cuantificación de las proteínas sinápticas es una técnica necesaria y útil para comprender mejor la forma en que las neuronas responden a estímulos y alterar la eficacia sináptica 7. Este artículo describe una técnica común para aislar proteínas sinápticas de tejido cerebral de roedor por ultracentrifugación en gradientes de sacarosa discontinuos. La fracción de membrana plasmática sináptica puede ser enriquecido y aislado basándose en elsu densidad en sacarosa, que ha sido determinado empíricamente para ser similar a 1.2 M de sacarosa. Dependiendo de la pregunta biológica, fracciones subcelulares se pueden separar por gradientes continuos o discontinuos de Percoll, ya sea sacarosa o. Gradientes continuos permiten la separación de las proteínas en fracciones múltiples; esto puede ser particularmente útil para demostrar la co-localización de las proteínas dentro de una fracción dada 8. Sin embargo, la preparación de gradientes continuos es más laborioso y no es necesaria para muchas aplicaciones. Gradientes discontinuos son comparativamente más fáciles de preparar y se pueden utilizar para separar proteínas en unas pocas fracciones, definidos en general-. Gradientes discontinuos que están compuestos de tres capas de sacarosa de molaridad creciente han sido ampliamente utilizados para aislar proteínas asociadas con la membrana plasmática sináptica (SPM). Esta fracción de membrana plasmática sináptica se puede procesar adicionalmente a la fractio densidad post-sináptican (PSD) por tratamiento con detergente y el aislamiento de la fracción insoluble en detergente. Cuando este proceso fue descrito por primera vez en el 1960 de 9,10, se utilizó microscopía electrónica para demostrar los orgánulos y membranas que definen aproximadamente la membrana plasmática sináptica y fracciones de densidad post-sináptica 9-14. Estos estudios demostraron la inclusión de membranas pre y post-sinápticas y vesículas sinápticas en la fracción SPM; después de tratamiento con detergente principalmente el electrón-densos, las densidades de post-sinápticas eran visibles. En el procedimiento, un choque hipotónico se utiliza para evitar el paso de los procesos sinápticos desde el cuerpo celular 10. Este paso se aprovecha del hecho de que las mitocondrias son más resistentes a choque osmótico y permanecen intactos, y por lo que se sedimentan en la parte inferior del gradiente de sacarosa (Figura 1). Utilizando esta misma técnica de enriquecimiento, las fracciones de SPM y PSD fueron primero bioquímicamentedefinido por electroforesis en gel de poliacrilamida y la secuenciación de los principales componentes de la proteína de 15-17. El análisis de transferencia Western Posteriormente se ha utilizado para detectar y cuantificar los niveles de proteínas sinápticas y definir estas fracciones (Figura 2). Hemos utilizado esta técnica en nuestros laboratorios para cuantificar los cambios en los niveles sinápticos de transportador de dopamina que se producen cuando el locus SLC6A3 se duplica en ratones 18. También hemos utilizado esta técnica en ratones deficientes del receptor NMDA para descubrir reducciones sinápticos específicos en las proteínas que forman parte de la interactome DISC1 19. Es evidente a partir de análisis de transferencia Western que las fracciones SPM contienen proteínas sinápticas membrana de la vesícula, marcadores endosoma, proteínas asociadas a la membrana mitocondrial, enzimas sintéticas y moléculas de transducción de señales, así como componentes integrales de la densidad post-sináptica y las membranas plasmáticas sinápticas 20-23. Efracciones PSD VEN pueden tener contaminación con abundantes proteínas mitocondriales y puede ser necesario realizar una segunda sedimentación gradiente o etapas de purificación adicionales para eliminarlos 13. Recientemente, la espectrometría de masas cuantitativa ha proporcionado una lista de más de 100 proteínas en la densidad post-sináptica solo, así como una indicación de la abundancia relativa de estos componentes 24,25.

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preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

Técnicas de fraccionamiento subcelular neuronales permiten la cuantificación de proteínas que son objeto de trata hacia y desde la sinapsis. Como se describió originalmente en la década de 1960, las proteínas asociadas con la membrana plasmática sináptica se pueden aislar mediante ultracentrifugación en un gradiente de densidad de sacarosa. Una vez que se aislaron las membranas sinápticas, el complejo macromolecular conocido como la densidad post-sináptica puede ser posteriormente aislado debido a su insolubilidad detergente. Las técnicas usadas para aislar las membranas plasmáticas sinápticas y proteínas de densidad post-sinápticas siguen siendo esencialmente la misma después de 40 años, y se utilizan ampliamente en la investigación neurocientífica actual. Este artículo detalla el fraccionamiento de las proteínas asociadas con la membrana plasmática sináptica y post-sináptica densidad utilizando un gradiente discontinuo de sacarosa. Preparaciones de proteínas resultantes son adecuadas para el Western Blot o análisis DIGE 2D.

La preparación del gradiente de densidad de sacarosa debe dar lugar a una clara separación de las tres soluciones molares de sacarosa (0,8, 1,0, y 1,2 M de sacarosa). Véase la Figura 3A para un ejemplo del gradiente antes de añadir la muestra de proteína. Si el gradiente se prepara con demasiada antelación, o si se prepara en una superficie de la mesa con la vibración de otro equipo, el gradiente se verá comprometida y no se logra una separación adecuada. Si una clara separación de las tres so...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Este artículo detalla el enriquecimiento de las proteínas asociadas con la membrana plasmática sináptica por ultracentrifugación en un gradiente discontinuo de sacarosa. También se describe la preparación posterior de las proteínas de densidad post-sináptica. Preparaciones de proteínas son adecuados para el Western Blot o análisis DIGE 2D.

Preparación de sináptica Membrana plasmática y proteínas postsináptica densidad utilizando un gradiente discontinuo de sacarosa

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

31.0K vistas.  University of Toronto.  Este artículo detalla el enriquecimiento de las proteínas asociadas con la membrana plasmática sináptica por ultracentrifugación en un gradiente discontinuo de sacarosa. También se describe la preparación posterior de las proteínas de densidad post-sináptica. Preparaciones de proteínas son adecuados para el Western Blot o análisis DIGE 2D. 

Video: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

El estudio de Piscinas vesícula sináptica con fotoconversión de tintes estirilo

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Investigación

Neurociencias

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

Los potenciales de membrana, las respuestas sinápticas, circuitos neuronales, neuromodulación y la histología muscular utilizando el cangrejo de río: Los ejercicios de laboratorio para estudiantes

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Preparación de Synaptoneurosomes de ratón con una corteza discontinua de Percoll-sacarosa gradiente de densidad

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un método general para la evaluación de incubación de sacarosa en ratas Deseo

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusión lateral y exocitosis de proteínas de membrana en neuronas cultivadas evaluó a través de la recuperación de fluorescencia y la pérdida de fluorescencia photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Post-Etiquetado Inmunoelectrónica incorporación de proteínas sinápticas en cultivos de cortes de hipocampo

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivado de la evolución temporal de la Liquidación glutamato con un análisis de deconvolución de Corrientes Transporter astrocíticos

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

La unión neuromuscular: Medición Synapse Tamaño, fragmentación y los cambios en la densidad de proteína sináptica Uso confocal microscopía de fluorescencia

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.