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
发布: 2015年02月20日第5卷第4期 DOI: 10.21769/BioProtoc.1398 浏览次数: 14680
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:
Dec 2013
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
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
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.
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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.
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分类
神经科学 > 行为神经科学 > 学习和记忆
生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 定量
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