从经过运动技能培训的小鼠的运动皮质中制备突触体

Anand Suresh; Anna Dunaevsky

doi:10.21769/BioProtoc.1398

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Peer-reviewed

Preparation of Synaptosomes from the Motor Cortex of Motor Skill Trained Mice

从经过运动技能培训的小鼠的运动皮质中制备突触体

Anand Suresh

Anna Dunaevsky email

发布: 2015年02月20日第5卷第4期 DOI: 10.21769/BioProtoc.1398 浏览次数: 14630

评审: Masahiro MoritaSoyun KimPamela Maher

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Dec 2013

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Abstract

Learning and memory are thought to occur due to changes in synaptic strength. Strengthening of synapses due to Long Term Potentiation mechanisms are mediated by increases in synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) levels. Here we describe a protocol to isolate and quantify synaptic AMPAR subunit GluA1 levels from the motor cortex of mice which have undergone motor skill training.

Keywords: Synaptosome (突触小体)

Motor cortex (运动皮层)

Slices (片)

Materials and Reagents

C57BL6 mouse (Mus musculus)
Isoflurane (Isothesia) (Butler animal supplies)
Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: S-271 )
Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: P217-500 )
Sodium bicarbonate (NaHCO₃) (Thermo Fisher Scientific, catalog number: S233-500 )
Monosodium phosphate (NaH₂PO₄) (Thermo Fisher Scientific, catalog number: S369-500 )
Magnesium sulphate heptahydrate (MgSO₄^.7H₂O) (Thermo Fisher Scientific, catalog number: MG63-500 )
Calcium chloride (CaCl₂) (Thermo Fisher Scientific, catalog number: C79-500 )
Dextrose (Thermo Fisher Scientific, catalog number: D16-500 )
Sucrose (Thermo Fisher Scientific, catalog number: S5-3 )
Radio immunoprecipitation assay (RIPA) lysis buffer (see Recipes)
Roche complete protease inhibitor cocktail pellets (Roche Diagnostics, catalog number: 11697498 001 )
Protease inhibitor (Sigma-Aldrich, catalog number: p8340 )
Phosphatase inhibitor (Sigma-Aldrich, catalog number: 3 p0044 )
Krazy glue
Millipore anti GluA1 antibody(Millipore, catalog number: ab1504 )
Cell signaling anti-rabbit HRP antibody (Cell Signaling Technology, catalog number: 7074S )
Agar powder (Alfa Aesar, catalog number: A10752 )
Artificial cerebrospinal fluid (ASCF) (see Recipes)
High magnesium ACSF (see Recipes)
Agar block (see Recipes)
Sucrose media (see Recipes)

Equipment

Leica vibratome S1000
Scissors (one large and one small)
Forceps
Sharp blade
Vibratome injector blade (Leica)
1.5 ml Eppendorf tube
Pellet pestle motor hand held homogenizer (Kontes, catalog number: 749540-0000 )
4 ºC table top centrifuge
Ruler
Aerobic air mixture (Lindweld Alloy, model: MAA140 )
Square petri dish with Grid (Thermo Fisher Scientific, catalog number: FB0875711A )
Sonicator (Thermo Fisher Scientific)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Suresh, A. and Dunaevsky, A. (2015). Preparation of Synaptosomes from the Motor Cortex of Motor Skill Trained Mice. Bio-protocol 5(4): e1398. DOI: 10.21769/BioProtoc.1398.
Padmashri, R., Reiner, B. C., Suresh, A., Spartz, E. and Dunaevsky, A. (2013). Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome. J Neurosci 33(50): 19715-19723.