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Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO)
从大鼠正中视前核(MnPO)中制备原代神经元   

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Abstract

Studying cell physiology is really important to understand their function and to determine action mechanisms taking place in these cells. Using brain slices can be sometime difficult to directly access to cells like neurons. Even if it more steps are needed to get cultured cells, this type of preparation allow a better access to neurons and provide a good way to study their basal properties. Here we describe a protocol of a short term primary neurons culture from MnPO, allowing to kept neurons in good condition to perform electrophysiological recordings.

Materials and Reagents

  1. Wistar rats (3 weeks old) (Charles River Laboratories International)
  2. Ketamine-xylasine mixture
  3. Pronase (Sigma-Aldrich, catalog number: 9036-06-0 )
  4. Thermolysin (Sigma-Aldrich, catalog number: 9073-78-3 )
  5. Bovine Serum Albumin (BSA) (Life Technologies, catalog number: 15561020 )
  6. Laminin (Sigma-Aldrich, catalog number: 114956-81-9 )
  7. Ruthenium red
  8. KCl (Sigma-Aldrich, catalogue number: 7447-40-7 )
  9. CaCl2 (Sigma-Aldrich, catalogue number: 10043-52-4 )
  10. MgCl2 (Sigma-Aldrich, catalogue number: 7786-30-3 )
  11. NaHCO3 (Sigma-Aldrich, catalogue number: 144-55-8 )
  12. NaH2PO4 (Sigma-Aldrich, catalogue number: 7558-80-7 )
  13. NaCl (Sigma-Aldrich, catalogue number: 7647-14-5 )
  14. Sucrose (Sigma-Aldrich, catalogue number: 57-50-1 )
  15. HEPES (Sigma-Aldrich, catalogue number: 7365-45-9 )
  16. D-Glucose (Sigma-Aldrich, catalogue number: 50-99-7 )
  17. Dissection solution (see Recipes)
  18. Artificial Cerebrospinal Fluid (aCSF) (see Recipes)

Equipment

  1. Bath
  2. Vibratome
  3. 0.45 μm sterilize filter
  4. 350 μm Sagittal slice containing MnPO
  5. Needle
  6. Pasteur pipettes
  7. Centrifuge
  8. 37 °C, 95% O2-5% CO2 Cell culture incubator
  9. 6 hole petri dish (Thermo Fisher Scientific, catalog number: 50-341-84 )
  10. Micro-cover glasses (Thermo Fisher Scientific, catalogue number: NC9216450 )
  11. 15 ml Falcon tubes
  12. Oxygen and Carbogen tank
  13. Binocular

Procedure

  1. Micro-cover glass is coated with laminin (5 μg/ml) and incubated at 37 °C, 95% O2-5% CO2 Cell culture incubator, 3 h before culture preparation.
  2. Wistar rats is deeply anesthetized by intraperitoneal injection of a ketamine-xylasine mixture (87.5 and 12.5 ml/kg, respectively) and decapitated.
  3. The brain is removed from the skull and immersed in oxygenated (95% O2-5% CO2) ice-cold (2 °C) dissection solution. Oxygenation system consist on a 95% O2-5% CO2 tank connected to a bath with a line transmitting a gentle bubbling. Then the brain is fixed with glue on a plate which will be place in the Vibratome tub containing oxygenated dissection solution.
  4. A sagittal slice of 350 μm containing the MnPO is prepared using Vibratome (Figure 1).


    Figure 1. Use Vibratome to do brain slices

  5. Using binocular, the ventral region of the MnPO is punched (about 3mm in diameter) out with a curve needle (Figure 2), and placed in 1 ml aCSF solution (in falcon) containing 0.1 mg/ml of pronase for 10 min at 37 °C and oxygenated.
    Note: Curve needle is homemade from an aluminum needle tip 3 mm diameter, gently curved at 2/3 of length with a soldering and fixed to a 10 ml syringe.

     
    Figure 2. Use Binocular to realize MnPO micropunch

  6. Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 2 mg/ml of BSA for 15 min at 37 °C and oxygenated.
  7. Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 0.1 mg/ml of thermolysin for 10 min at 37 °C and oxygenated.
  8. Transfer solution containing tissue in a 1.5 ml Eppendorf tube.
  9. The tissue is then mechanically dissociated by successive trituration with glass Pasteur pipettes (Figure 3).
    Note: Use 4 Pasteur pipettes whose diameter has previously been gradually reduced with flame from no change to 50 μm. This step is done at room temperature. The come and go flow should be strong enough to separate cells, but also enough gentle to not break cell membrane. The size of micropunch should reduce from pipette to another, and completely disappear using the last pipette.

     
    Figure 3. Mechanic trituration with Pasteur pipette

  10. Transfer solution containing cells in a 1.5 ml Eppendorf tube.
  11. Solution containing cell is centrifuged at 1500 x g during 2 min at room temperature.
    Note: Sometime pellet is really small and difficult to see, but there are cells on the wall of the tube.
  12. The supernatant is removed and cells are re-suspended in 50 μl aCSF.
    Note: Slow agitation for re-suspension to not break the membranes.
  13. The aCSF solution containing cells (50 μl) is directly platted on micro cover glasses beforehand treated with laminine, then disposed in 6 holes petri dish, and incubated 1 h at 37 °C, 95% O2-5% CO2, before patch-clamp recording.
    Notes:
    1. There is no culture media, cells remains during 1 h in aCSF solution. Do not exceed 1 h of incubation. Density of cell is relatively low, about 100 cells on 1 micro cover glass.
    2. Be sure that the solution stays on the micro-cover glass in bead, if not preparation will be dry and unusable.

Recipes

  1. Dissection solution
    200 mM sucrose
    10 mM D-Glucose
    2 mM KCl
    1 mM CaCl2
    3 mM MgCl2
    26 mM NaHCO3
    1.25 mM NaH2PO4, pH 7.4
    Adjusted with CO2 bubbling and verified with Ruthenium red
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  2. Artificial cerebrospinal fluid/aCSF
    140 mM NaCl
    3.1 mM KCl
    2.4 mM CaCl2
    1.3 mM MgCl2
    10 mM HEPES
    10 mM D-Glucose, pH 7.4 adjusted with KOH, osmolarity 300 MOsm
    Filter sterilize (0.45 μm)
    Stored at 4 °C

Acknowledgments

This protocol was adapted from: Tremblay et al. (2011). This work was supported by Canadian Institutes of Health Research Grant MOP-178002.

References

  1. Berret, E., Nehme, B., Henry, M., Toth, K., Drolet, G. and Mouginot, D. (2013). Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population. J Neurosci 33(7): 3067-3078.
  2. Tremblay, C., Berret, E., Henry, M., Nehme, B., Nadeau, L. and Mouginot, D. (2011). Neuronal sodium leak channel is responsible for the detection of sodium in the rat median preoptic nucleus. J Neurophysiol 105(2): 650-660.

简介

研究细胞生理学对于了解其功能和确定在这些细胞中发生的作用机制是非常重要的。 使用脑切片可能有时难以直接访问像神经元这样的细胞。 即使需要更多的步骤来培养细胞,这种类型的制剂可以更好地接近神经元,并提供研究其基础性质的好方法。 在这里,我们描述了一种来自MnPO的短期原代神经元培养的方案,允许神经元保持良好的状态进行电生理记录。

材料和试剂

  1. Wistar大鼠(3周龄)(Charles River Laboratories International)
  2. 氯胺 - 二甲苯胺混合物
  3. Pronase(Sigma-Aldrich,目录号:9036-06-0)
  4. Thermolysin(Sigma-Aldrich,目录号:9073-78-3)
  5. 牛血清白蛋白(BSA)(Life Technologies,目录号:15561020)
  6. 层粘连蛋白(Sigma-Aldrich,目录号:114956-81-9)
  7. 钌红
  8. KCl(Sigma-Aldrich,目录号:7447-40-7)
  9. CaCl 2(Sigma-Aldrich,目录号:10043-52-4)
  10. MgCl 2(Sigma-Aldrich,目录号:7786-30-3)
  11. NaHCO 3(Sigma-Aldrich,目录号:144-55-8)
  12. NaH 2 PO 4(Sigma-Aldrich,目录号:7558-80-7)
  13. NaCl(Sigma-Aldrich,目录号:7647-14-5)
  14. 蔗糖(Sigma-Aldrich,目录号:57-50-1)
  15. HEPES(Sigma-Aldrich,目录号:7365-45-9)
  16. D-葡萄糖(Sigma-Aldrich,目录号:50-99-7)
  17. 解剖溶液(见配方)
  18. 人工脑脊液(aCSF)(见配方)

设备


  1. Vibratome
  2. 0.45μm灭菌过滤器
  3. 350μm含MnPO的矢状切片

  4. 巴斯德移液器
  5. 离心机
  6. 37℃,95%O 2 -5%CO 2细胞培养孵化器
  7. 6孔培养皿(Thermo Fisher Scientific,目录号:50-341-84)
  8. 微盖玻璃(Thermo Fisher Scientific,目录号:NC9216450)
  9. 15 ml Falcon管
  10. 氧气和碳水箱
  11. 双目

程序

  1. 微量盖玻片用层粘连蛋白(5μg/ml)包被,并在37℃,95%O 2 -5%CO 2细胞培养箱中温育3小时 培养物制备前。
  2. 通过腹膜内注射氯胺酮 - 赛拉嗪混合物(分别为87.5和12.5ml/kg)并且断头,将Wistar大鼠深度麻醉。
  3. 从颅骨中取出脑,并浸入含氧的(95%O 2 -5%CO 2)冰冷(2℃)解剖溶液中。氧合系统由连接到具有传送轻微起泡的管线的浴的95%O 2 -5%CO 2罐组成。然后将大脑用胶固定在板上,该板将放置在含有氧合解剖溶液的Vibratome浴缸中。
  4. 使用Vibratome(图1)制备含有MnPO的矢状切片350μm

    图1.使用Vibratome做脑切片

  5. 使用双眼,用曲线针(图2)冲出MnPO的腹侧区域(直径约3mm),并在37℃下放置在含有0.1mg/ml链霉蛋白酶的1ml aCSF溶液(在falcon中)中10分钟°C并充氧。
    注意:曲线针是用直径3毫米的铝针尖自制的,用焊接方法以2/3的长度轻轻弯曲并固定到10毫升注射器。

     
    图2.使用双眼实现MnPO微销

  6. 将Micropunch转移到含有2mg/ml BSA的1ml aCSF溶液的新falcon管中,在37℃下15分钟并充氧。
  7. 将微穿孔转移到含有0.1mg/ml嗜热菌蛋白酶的1mlαCSF溶液的新falcon管中,在37℃下氧化10分钟。
  8. 在1.5ml Eppendorf管中含有组织的转移溶液。
  9. 然后通过用玻璃巴斯德移液管连续研磨将组织机械解离(图3) 注意:使用4个巴斯德移液管,其直径已经逐渐减少,火焰从没有变化到50微米。该步骤在室温下进行。来来去流应该足够强,以分离细胞,但也足够温和,以不打破细胞膜。微量注射的大小应该从移液器减少到另一个,并使用最后一个移液管完全消失。

     
    图3.使用巴斯德吸管的机械研磨

  10. 含有细胞的转移溶液在1.5ml Eppendorf管中。
  11. 含有细胞的溶液在室温下以1500×g离心2分钟 注意:有时颗粒很小,很难看到,但管壁上有细胞。
  12. 除去上清液,将细胞重悬于50μlaCSF中 注意:缓慢搅拌以重新悬浮以不破坏膜。
  13. 将含有细胞(50μl)的aCSF溶液直接置于预先用层粘连蛋白处理的微覆盖玻璃上,然后置于6孔培养皿中,并在37℃,95%O 2 - 5%CO 2 2,在膜片钳记录之前。
    注意:
    1. 没有培养基,细胞在aCSF溶液中保持1小时。不要超过1小时的孵化。细胞密度相对较低,在1微盖玻片上约100个细胞。
    2. 确保溶液保持在微覆盖玻璃珠上,如果不是准备将干燥和不能使用。

食谱

  1. 解剖溶液
    200mM蔗糖 10 mM D-葡萄糖
    2 mM KCl
    1mM CaCl 2
    3mM MgCl 2/
    26mM NaHCO 3/v/v 1.25mM NaH 2 PO 4,pH7.4 用CO 2鼓泡调节并用钌红
    验证 过滤灭菌(0.45μm)
    储存在4°C
  2. 人工脑脊液/aCSF
    140mM NaCl 3.1 mM KCl
    2.4mM CaCl 2 1.3mM MgCl 2·h/v 10 mM HEPES
    10mM D-葡萄糖,用KOH调节pH7.4,渗透压为300MWsm 过滤灭菌(0.45μm)
    储存在4°C

致谢

该协议改编自:Tremblay等人(2011)。 这项工作得到加拿大卫生研究院研究所MOP-178002的支持。

参考文献

  1. Berret,E.,Nehme,B.,Henry,M.,Toth,K.,Drolet,G。和Mouginot,D。 中心Na + 检测的调节需要NaX通道的协同作用 和α1同种型Na + /K + -ATPase。 J Neurosci 33(7):3067-3078。
  2. Tremblay,C.,Berret,E.,Henry,M.,Nehme,B.,Nadeau,L.和Mouginot,D。(2011)。神经元钠泄漏通道负责检测大鼠中央前视核中的钠。 Neurophysiol 105(2):650-660。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Berret, E. (2013). Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO). Bio-protocol 3(23): e984. DOI: 10.21769/BioProtoc.984.
  2. Berret, E., Nehme, B., Henry, M., Toth, K., Drolet, G. and Mouginot, D. (2013). Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population. J Neurosci 33(7): 3067-3078.
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