JoVE Journal > Neuroscience
Summary
Abstract
Protocol
Discussion
Disclosures
Acknowledgements
Materials
References
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神经科学

Preparação de Synaptoneurosomes de rato Cortex usando um gradiente de densidade Percoll descontínuo Sacarose-

Published: September 17, 2011
doi:
10.3791/3196
, , ,
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities,University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities,University of Wisconsin

Summary

Um método para preparar traducionalmente ativa, synaptoneurosomes intacta (SNs) de córtex cerebral de rato é descrito. O método utiliza um gradiente de densidade descontínua Percoll-sacarose permitindo a preparação rápida de ativos SNs.

Abstract

Synaptoneurosomes (SNS) são obtidos após homogeneização e fracionamento de rato córtex cerebral. Eles são selados vesículas ou terminais isolados que romper com os terminais do axônio quando o tecido cortical é homogeneizado. Os SNs reter características pré e pós-sináptico, o que os torna útil no estudo da transmissão sináptica. Conservem as máquinas moleculares utilizados na sinalização neuronal e são capazes de captação, armazenamento e liberação de neurotransmissores.

A produção e isolamento de ativos SNs pode ser problemático mídias usando como Ficoll, que podem ser citotóxicos e centrifugação prolongada devido a alta densidade, e filtração e métodos de centrifugação, que pode resultar em baixa atividade, devido a danos mecânicos do SNS. No entanto, o uso de gradientes descontínuos de Percoll-sacarose de densidade para isolar SNs fornece um método rápido para produzir bons rendimentos de SNs traducionalmente ativo. O método do gradiente Percoll-sacarose é rápido e gentil como ele emprega condições isotônica, tem spins centrifugação menos e mais curtos e evita que as etapas de centrifugação pellet SNs e causar danos mecânicos.

Protocol

1. Preparações 1-4 Prepare 500 mL de tampão de meio de gradiente (GM), misturando 50 mL de uma pasta de sacarose 2,5 M, 2,5 mL de uma 1M Tris-HCl, pH 7,5 valores, e 0,1 mL de um 0,5 m EDTA, pH 8,0 estoque com mili- Q água para volume. Filtro de esterilizar a solução de alíquota, e armazenar congelados. Prepare um estoque de 1000x tetrodotoxina (TTX) fazendo uma solução de 1 mM em mili-Q H 2 O. Alíquotas de TTX podem ser armazenadas a -20 ° C. Preparar o tam…

Discussion

A preparação de gradiente descontínuo de Percoll-sacarose aqui descrito é um método rápido e confiável para isolar SNs ativo, que pode ser usado em uma variedade de experimentos de transmissão sináptica. Este método do gradiente, que é baseado no método desenvolvido por Dunkley et al., 3,4 é um procedimento de fracionamento subcelular cérebro que isola pré e pós-sináptica da membrana derivada vesículas que estão associados com o outro. Sem purificação adicional esses SNs são compatíveis…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Gostaríamos de agradecer a BK agosto da Universidade de Wisconsin-Madison Facility Microscópio Eletrônico para a microscopia eletrônica. Este trabalho foi financiado pelo NIH concede R01-DA026067 e P30-HD03352 (a JSM).

Materials

Name of the reagent Company Catalogue number Comments (optional)
Micro BCA Protein Assay Kit Pierce 23235  
CaCl2 Fisher C79-500  
CO2 gas Airgas (UW-MDS) CD 50  
EDTA RPI E57020  
EtOH Fisher A407SK-4  
HCl Fisher A142-212  
Percoll GE Healthcare 17-0891-01  
KH2PO4 Fisher P285-500  
PierceSDS-PAGE Sample Prep Kit Pierce 89888  
NaCl RPI S23020  
NaHCO3 Fisher BP328-500  
Na2PHO4 Fisher S381-500  
Sucrose RPI S24060  
Tris Base RPI T60040  
Tetrodotoxin Sigma T5651  
Express Pro Label Mix S35 Easy Tag Perkin Elmer NEG772  
       
Equipment Company Catalogue number Comments (optional)
Dissection tools      
Dounce homogenizer, 7 mL (comes with two glass pestles labled ” A” and “B”) Wheaton    
P1000 Gilson Pipetman Gilson F123602  
Allegra 6KR Centrifuge Beckman Coulter 366830  
GH 3.8 Rotor, Swinging bucket rotor Beckman Coulter 360581  
Beckman J2-21 Centrifuge Beckman    
Beckman tubes with caps Beckman 355672  
White walled adapters Beckman 342327  
Blue walled adapters Beckman    
JA-17 Rotor, Fixed-angle rotor Beckman 369691  

Table of antibodies used for western blots:

Name of Antibody Company Catalogue number Host Species Dilution Factor
β-Actin Sigma A5441 mouse 1:2000
GFAP Santa Cruz sc-65343 mouse 1:200
GP73 Santa Cruz Sc-134509 rabbit 1:200
HSC70 Santa Cruz sc-7298 mouse 1:200
Laminβ Santa Cruz Sc-6261 goat 1:200
Prohibitin Santa Cruz sc-28259 rabbit 1:200
PSD95 Millipore MAB1596 mouse 5 μg/μL
SNAP25 AbCam ab5666-100 rabbit 1:2000
Synaptophysin Millipore MAB368 mouse 1:500
β-3-Tubulin Santa Cruz sc-80016 mouse 1:200

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Tags

SynaptoneurosomesMouse CortexDiscontinuous Percoll-Sucrose Density GradientHomogenizationFractionationResealed VesiclesIsolated TerminalsSynaptic TransmissionPre- And Postsynaptic CharacteristicsNeuronal SignalingNeurotransmittersFicollCytotoxicCentrifugationFiltrationMechanical DamagePercoll-sucrose Gradient MethodTranslationally Active SNsIsotonic Conditions
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Westmark, P. R., Westmark, C. J., Jeevananthan, A., Malter, J. S. Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient. J. Vis. Exp. (55), e3196, doi:10.3791/3196 (2011).
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