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: 14913
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
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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
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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