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Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs)
大鼠原代胚胎基底前脑胆碱能神经元(BFCN)的分离与培养   

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Abstract

The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) is believed to be involved in many disorders of the brain such as Alzheimer’s disease (AD), schizophrenia, sleep disorders and drug abuse (Mobley et al., 1986). Primary cultures of BFCNs will provide an important tool for studying the mechanism of these diseases. This protocol provides a detailed description of experimental procedures in establishing in vitro primary culture of rat embryonic BFCNs.

Keywords: Basal forebrain cholinergic neuron(基底前脑胆碱能神经元), Nerve growth factor(神经生长因子), Alzheimer’s disease(阿尔茨海默氏病), in vitro culture(体外培养), Microfluidic chamber(微流控室), Axonal transport(轴突运输), Neurodegeneration (神经退变)

Background

The basal forebrain cholinergic system innervates the cerebral cortex and hippocampus. The normal function of the BFCNs is essential for normal sleeping, learning and memory. And the atrophy of BFCNs is considered as the early event of Alzheimer’s disease. Thus, the primary BFCNs culture will be the ideal cell model for AD research. In previous studies, primary BFCNs cultures were rarely used. Here, we present a reliable method to isolate and culture BFCNs from the embryonic rat septum which is simple, less time consuming than the previous method (Schnitzler et al., 2008). Our method will greatly facilitate studies of many critical aspects of BFCN function and cell biology.

Materials and Reagents

  1. Pipette tips
  2. Coverglasses (Thermo Fisher Scientific or VWR)
  3. Falcon tubes (50 ml)
  4. Falcon tubes (15 ml)
  5. Sterile pipettes 5, 10, 25 ml
  6. 12-well culture plate
  7. 10, 15 cm cell culture dishes
  8. Razor blades
  9. Fire-polished glass Pasteur pipettes

Note: Materials #3-9 can be from various suppliers.

  1. Microfluidic chamber (XONA MICROFLUIDICS, catalog number: SND450 )
  2. Stericup® filter units (150, 250 ml) (EMD Millipore, catalog number: SCVPU02RE , 0.10 µm, polyethersulfone)
  3. Pregnant female Sprague Dawley rats (Embryos dissected at E17.5) (Harlan Sprague Dawley)
  4. Poly-L-lysine 0.1% (w/v) in dH2O (Sigma-Aldrich, catalog number: P8920 )
  5. Phosphate-buffered saline (PBS) (without CaCl2, MgCl2) (Thermo Fisher Scientific, GibcoTM, catalog number: 1419094 )
  6. 70% ethanol
  7. 10x, 2.5% trypsin, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  8. 10x DNase I (10 mg/ml) (Roche Diagnostics, catalog number: 10104159001 )
  9. Anti-TrkA antibody
  10. 10x Hanks’ balanced salt solution (HBSS) w/o PR, sod.bicarb, CaMg (Mediatech, catalog number: 20-023-CV )
  11. Penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  12. Neurobasal-A medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
  13. Fetal bovine serum (FBS) (Qmega Scientific, catalog number: FB-02 or various suppliers) (heat in activation before use)
  14. 50x B27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  15. 100x GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  16. Mouse purified NGF as previously published (Mobley et al., 1986)
  17. Dissection buffer (see Recipes)
  18. Plating media (see Recipes)
  19. Maintenance media (see Recipes)

Equipment

  1. Sterile cell culture hood (Esco Micro, model: Class II Type A2 )
  2. Scissors
  3. #5 biologie forces and small spring scissors (Fine Science Tools)
  4. Pipette
  5. Water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: PrecisionTM Model 281 )
    Note: This product has been discontinued.
  6. Cell culture incubator (Panasonic, model: MCO-19AIC UV-PA )
  7. Dissecting microscope (Leica, model: Leica S8 AP0 )
  8. Cell culture centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: CL2 )
  9. Autoclave (STERIS, model: AMSCO® Evolution® Steam Sterilizer )

Procedure

  1. Preparation
    1. Coat plates/coverglasses using poly-L-lysine (PLL) for 12 h at 4 °C or for 2-4 h at 37 °C. The coverglasses are immersed in PLL in a 10 cm cell culture plate. For coating 12-well culture plates, 0.5 ml of PLL is added to each well to cover the whole surface.
    2. Wash 3-4 x with 1 ml cold PBS followed by a quick rinse with 1 ml sterile water, and air dry the plate or coverglasses in the sterile cell culture hood.
    3. Disinfect all dissection tools in 70% ethanol.

  2. Embryo dissections (all procedures have been approved by UCSD IACUC)
    One Sprague Dawley female pregnant rat (E17.5)
    1. Anesthetize the rat in an approved CO2 chamber.
    2. Perform cervical dislocation.
    3. Disinfect the rat’s abdomen with 70% ethanol.
    4. Cut the skin with scissors in a T fashion, posterior to anterior direction followed by an intersecting cut right above the genital area.
    5. Cut the muscle tissue in a similar pattern.
    6. Locate the horns of the uterus and sever with scissors, remove the uterus.
    7. Place uterus in a 10 cm TC dish and cut along the uterus freeing each embryo in its amniotic sac and placenta.
    8. Remove each embryo from its amniotic sac and transfer to a 10 cm TC dish with 10-15 ml 1x HBSS to cover all embryos. Place this dish on ice.
    Note: For steps B3-B8, Please see an excellent video presentation in Pacifici and Peruzzi (2012).

  3. BFCNs dissections
    1. Place an embryo into a 10 cm TC dish on ice. And the following dissection procedures are performed either at room temperature or on ice.
    2. Remove the head with a razor blade.
    3. Use #5 biologie forceps to cut the skin and soft bones to expose the whole brain.
    4. Gently extract the brain and put it on a 10 cm TC dish.
    5. Cut and remove the olfactory tubercles, cerebellum and brain stem.
    6. Carefully remove the meninges membrane (meninges will become separate from the brain and become more visible when the tissue is immersed in 1x HBSS, starting from the edge of the tissue, use forceps to carefully peel the reddish membrane off the brain, as complete as possible).
    7. The basal forebrain is just located close to the medial and ventral surfaces of the cerebral hemispheres.
    8. Gently open the cerebral hemispheres along the sagittal suture to expose the diencephalon and the basal forebrain. 
    9. Make a transversal cut to separate the posterior side of the basal forebrain from the anterior pole of the diencephalon.
    Note: For steps C1-C9, please refer to excellent photo and video presentations in Schnitzler et al., 2008. 
    1. Place the basal forebrain tissues in a 15 ml tube with 1x HBSS and place the tube in ice.
    2. Repeat steps C1-C9 and collect all the BFCN tissues.

  4. Mechanical/enzymatic dissociation of BFCNs
    1. Wash the BFCN tissues with 5 ml cold 1x HBSS, repeat for 4 times.
    2. Remove the buffer by pipette.
    3. Add 1.8 ml 1x HBSS, 0.2 ml 10x trypsin (2.5%).
    4. Incubate for 15 min in a 37 °C water bath.
    5. Add 0.2 ml 10x DNase (10 mg/ml) and gently pipette up and down 10-15 times with a fire-polished glass Pasteur pipette until the mixture becomes homogenous suspension with no visible undigested tissues.
    6. Pre-warm plating media at 37 °C before use.
    7. Add 5 ml pre-warmed plating media and spin for 5 min at 500 x g (or 1,000 rpm) at room temperature.
    8. Remove supernatant.
    9. Add plating media (volume determined by desired plating density/method).
      Note: 500 µl per well for 12-well plate or 150 µl for coverglass.
    10. After 30 min, add 1 ml plating media and put into a 37 °C CO2 incubator.
    11. Change to maintenance media the next day. The cultures can be maintained for 10-14 days with half the media changed to fresh media every other day.

Data analysis

Neurons can be cultured in microfluidic chamber (Figure 1A, left panel: a DIC image; right panel: an image of immunostaining with anti-TrkA antibody) to separate neuronal soma from long axons to study axonal transport. Neurons can also be grown in mass culture on coated coverglasses for other applications. For instance, immunostaining was used to confirm the presence of cholinergic (at DIV7), NGF-responsive neurons in the culture with specific antibodies against choline acetyltransferase (ChAT), a marker for cholinergic neurons, and TrkA, the receptor for NGF (Figure 1B). The majority of neurons (> 90%) were positive for both markers (Xu et al., 2016).


Figure 1. Cultures of rat E18 basal forebrain cholinergic neurons (BFCNs). A. Representative images of primary BFCNs (DIV7) (left: DIC; right: anti-TrkA immunostaining) cultured in microfluidic chamber. Scale bars = 50 μm. B. Representative images of primary BFCNs (DIV7) were costained for the cholinergic neuronal marker ChAT (red) and the NGF receptor TrkA (green). Scale bars = 10 μm.

Note: For representative photos and results please refer to (Xu et al., 2016).

Recipes

  1. Dissection buffer (filter sterilized)
    10 ml 10x Hanks’ balanced salt solution (HBSS)
    89 ml ddH2O
    1 ml Pen/Strep
  2. Plating media (filter sterilized)
    43.5 ml Neurobasal media
    5 ml FBS
    1 ml 50x B27
    0.5 ml 100x GlutaMAX
    3. Maintenance media
    Plating media plus 50 ng/ml NGF (final concentration)

Acknowledgments

This protocol was adapted from methods described by Schnitzler et al. (2008). The study is supported by the following grants: NIH (PN2EY016525), NIH UCSD ADRC P50 Pilot grant, Down Syndrome Research and Treatment Foundation, Larry L. Hillblom Foundation, Tau Consortium, the Ministry of Science and Technology of the People’s Republic of China (2014CB965002, 2012BAI10B03), National Natural Science Foundation of China (81171200), Science and Technology Commission of Shanghai Municipality (13JC1401502, 13140904000), Shanghai Municipal Education Commission (12ZZ115).

References

  1. Mobley, W. C., Rutkowski, J. L., Tennekoon, G. I., Gemski, J., Buchanan, K. and Johnston, M. V. (1986). Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons. Brain Res 387(1): 53-62.
  2. Pacifici, M. and Peruzzi, F. (2012). Isolation and culture of rat embryonic neural cells: a quick protocol. J Vis Exp (63): e3965.
  3. Schnitzler, A. C., Lopez-Coviella, I. and Blusztajn, J. K. (2008). Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons. Nat Protoc 3(1): 34-40.
  4. Xu, W., Weissmiller, A. M., White, J. A., 2nd, Fang, F., Wang, X., Wu, Y., Pearn, M. L., Zhao, X., Sawa, M., Chen, S., Gunawardena, S., Ding, J., Mobley, W. C. and Wu, C. (2016). Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration. J Clin Invest 126(5): 1815-1833.

简介

基底前脑靠近从小脑发育的大脑半球的内侧和腹侧表面。 它调节多个过程,包括注意力,学习,记忆和睡眠。 据信基础前脑胆碱能神经元(BFCNs)的功能障碍和退化参与许多脑部疾病,如阿尔茨海默病(AD),精神分裂症,睡眠障碍和药物滥用(Mobley等人,1986年)。 BFCN的主要文化将为研究这些疾病的机制提供重要的工具。 该方案提供了建立大鼠胚胎BFCN的体外原代培养的实验程序的详细描述。
【背景】基础前脑胆碱能系统支配大脑皮层和海马体。 BFCN的正常功能对于正常的睡眠,学习和记忆是非常重要的。 BFCN的萎缩被认为是阿尔茨海默病的早期事件。 因此,主要的BFCNs培养将是AD研究的理想细胞模型。 在以前的研究中,很少使用初级BFCN培养物。 在这里,我们提出了一种从胚胎大鼠隔膜中分离和培养BFCNs的可靠方法,该方法简单,比以前的方法耗时少(Schnitzler等人,2008)。 我们的方法将大大方便研究BFCN功能和细胞生物学的许多关键方面。

关键字:基底前脑胆碱能神经元, 神经生长因子, 阿尔茨海默氏病, 体外培养, 微流控室, 轴突运输, 神经退变

材料和试剂

  1. 移液器提示
  2. CoverGlasses(Thermo Fisher Scientific或VWR)
  3. Falcon管(50毫升)
  4. 猎鹰管(15毫升)
  5. 无菌移液器5,10,25 ml
  6. 12孔培养板
  7. 10,15厘米细胞培养皿
  8. 剃刀刀片
  9. 火抛光玻璃巴斯德移液器

注意:材料#3-9可以来自各种供应商。

  1. 微流控室(XONA MICROFLUIDICS,目录号:SND450)
  2. 过滤单元(150,250ml)(EMD Millipore,目录号:SCVPU02RE,0.10μm,聚醚砜)
  3. 怀孕的雌性Sprague Dawley大鼠(胚胎在E17.5解剖)(Harlan Sprague Dawley)
  4. 在dH 2 O(Sigma-Aldrich,目录号:P8920)中的0.1%(w / v)的聚-L-赖氨酸
  5. 磷酸盐缓冲盐水(PBS)(不含CaCl 2,MgCl 2)(Thermo Fisher Scientific,Gibco< sup>目录号:1419094) br />
  6. 70%乙醇
  7. 10倍,2.5%胰蛋白酶,无酚红(Thermo Fisher Scientific,Gibco TM,目录号:15090046)
  8. 10x DNase I(10mg / ml)(Roche Diagnostics,目录号:10104159001)
  9. 抗TrkA抗体
  10. 10x汉克斯平衡盐溶液(HBSS)不含PR,sod.bicarb,CaMg(Mediatech,目录号:20-023-CV)
  11. 青霉素 - 链霉素(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  12. Neurobasal-A培养基(Thermo Fisher Scientific,Gibco TM,目录号:10888022)
  13. 胎牛血清(FBS)(Qmega Scientific,目录号:FB-02或各种供应商)(使用前激活加热)
  14. 50x B27补充剂(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  15. 100x GlutaMAX(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  16. 如先前公布的小鼠纯化的NGF(Mobley等人,1986)
  17. 解剖缓冲(见配方)
  18. 电镀介质(见配方)
  19. 维护媒体(见配方)

设备

  1. 无菌细胞培养罩(Esco Micro,型号:Class II Type A2)
  2. 剪刀
  3. #5生物制品力量和小弹簧剪刀(精细科学工具)
  4. 移液器
  5. 水浴(Thermo Fisher Scientific,Thermo Scientific TM,型号:Precision TM 型号281)
    注意:本产品已停产。
  6. 细胞培养箱(Panasonic,型号:MCO-19AIC UV-PA)
  7. 解剖显微镜(Leica,型号:Leica S8 AP0)
  8. 细胞培养离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:CL2)
  9. 高压釜(STERIS,型号:AMSCO ® Evolution ®蒸汽灭菌器)

程序

  1. 制备
    1. 在4℃下使用聚-L-赖氨酸(PLL)12小时的涂层板/覆盖层或在37℃下2-4小时。将覆盖层浸入10cm细胞培养板中的PLL中。为了涂覆12孔培养板,将0.5ml PLL加入到每个孔中以覆盖整个表面。
    2. 用1ml冷PBS洗涤3-4次,然后用1 ml无菌水快速冲洗,并在无菌细胞培养罩中空气干燥板或覆盖物。
    3. 消毒70%乙醇中的所有解剖工具
  2. 胚胎解剖(所有手术已获得UCSD IACUC批准)
    一只Sprague Dawley女性怀孕大鼠(E17.5)
    1. 在批准的CO 2 室麻醉大鼠。
    2. 执行宫颈脱位。
    3. 用70%乙醇消毒大鼠的腹部。
    4. 用剪刀以T形切割皮肤,在前方向后方,在生殖器区域正上方交叉切割。
    5. 以类似的模式切开肌肉组织。
    6. 找到子宫的角,用剪刀切割,取出子宫。
    7. 将子宫放置在10厘米TC盘中,沿子宫切割,使其羊膜和胎盘中的每个胚胎脱落。
    8. 从其羊膜囊中取出每个胚胎,并转移到10厘米的TC H皿中,以覆盖所有的胚胎。将这碟放在冰上。
    注意:对于B3-B8步骤,请参阅Pacifici和Peruzzi(2012)中的优秀视频演示。

  3. BFCN解剖
    1. 将胚胎置于冰上10厘米的TC盘中。并且在室温或冰上进行以下解剖手术。
    2. 用剃须刀刀头拆下头部。
    3. 使用#5生物镊子切割皮肤和软骨以暴露整个大脑。
    4. 轻轻取出大脑,放在10厘米TC盘上
    5. 切除并清除嗅结节,小脑和脑干。
    6. 小心地去除脑膜(脑膜会从脑部分开,当组织浸入1 HBSS时,从组织边缘开始变得更加明显,使用镊子,尽可能地完全从皮肤上剥离红色的薄膜。 )。
    7. 基底前脑恰好靠近大脑半球的内侧和腹侧。
    8. 沿着矢状缝线轻轻打开大脑半球以露出间脑和基底前脑。
    9. 进行横切,将基底前脑的后侧与间脑的前极分开。
    注意:对于步骤C1-C9,请参阅Schnitzler等人,2008年的优秀照片和视频演示。 
    1. 将基底前脑组织置于带有1 HBSS的15 ml管中,并将管置于冰中
    2. 重复步骤C1-C9并收集所有BFCN组织。

  4. BFCN的机械/酶分解
    1. 用5ml冷的1 HBSS洗涤BFCN组织,重复4次。
    2. 用移液器移除缓冲液。
    3. 加入1.8ml 1x HBSS,0.2ml 10x胰蛋白酶(2.5%)
    4. 在37°C水浴中孵育15分钟。
    5. 加入0.2 ml 10x DNA酶(10 mg / ml),用火抛光玻璃巴斯德移液管轻轻吸管10-15次,直到混合物变成均匀的悬浮液,没有可见的未消化的组织。
    6. 预热电镀介质在37°C使用前。
    7. 加入5ml预热的电镀介质,并在室温下以500×g(或1000rpm)旋转5分钟。
    8. 去除上清液
    9. 加入电镀介质(体积通过所需电镀密度/方法确定) 注意:对于12孔板,每孔500μl,或用于覆盖玻璃的150μl。
    10. 30分钟后,加入1ml电镀培养基,并放入37℃CO 2培养箱中。
    11. 第二天更换维护媒体。文化可以维持10-14天,其中一半的媒体每隔一天变成新鲜媒体。

数据分析

神经元可以在微流体室中培养(图1A,左图:DIC图像;右图:具有抗-TrkA抗体的免疫染色图像)以将神经元细胞从长轴突分离以研究轴突运输。神经元也可以用于其他应用的涂层覆盖物上的大量培养。例如,使用免疫染色来证实在具有抗胆碱乙酰转移酶(ChAT)的特异性抗体的培养物中胆碱能(DIV7),NGF反应性神经元的存在,胆碱能神经元的标记物和NGF的受体TrkA(图1B )。大多数神经元(> 90%)对于两种标记都是阳性的(Xu等人,2016)。


图1.大鼠E18基底前脑胆碱能神经元(BFCN)的培养物。 A.在微流体室中培养的原代BFCN(DIV7)(左:DIC;右:抗-TrkA免疫染色)的代表性图像。刻度棒=50μm。 B.胆碱能神经元标记物ChAT(红色)和NGF受体TrkA(绿色)的原代BFCN(DIV7)的代表性图像。比例尺=10μm
注意:对于代表性的照片和结果,请参阅(Xu et al。,2016)。

食谱

  1. 解剖缓冲液(过滤器灭菌)
    10毫克汉克斯平衡盐溶液(HBSS)
    89毫升ddH 2 O - / - 1 ml Pen / Strep
  2. 电镀介质(过滤器灭菌)
    43.5毫升Neurobasal媒体
    5 ml FBS
    1 ml 50x B27
    0.5ml 100x GlutaMAX
    维护媒体
    电镀培养基加50ng / ml NGF(终浓度)

致谢

该方案根据Schnitzler等人描述的方法进行了改进。 (2008年)。该研究得到以下资助:NIH(PN2EY016525),NIH UCSD ADRC P50试点奖学金,唐氏综合症研究和治疗基金会,Larry L. Hillblom基金会,中华人民共和国科技部Tau联盟( 2014CB965002,2012BAI10B03),中国国家自然科学基金(81171200),上海市科学技术委员会(13JC1401502,131340904000),上海市教委(12ZZ115)。

参考

  1. Mobley,WC,Rutkowski,JL,Tennekoon,GI,Gemski,J.,Buchanan,K.and Johnston,MV(1986)。< a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 3742234“target =”_ blank“>神经生长因子增加胆碱乙酰转移酶在发育基础前脑神经元中的活性。 386(1):53-
  2. Pacifici,M.和Peruzzi,F。(2012)。大鼠胚胎神经细胞的分离和培养:快速方案。
  3. Schnitzler,AC,Lopez-Coviella,I. and Blusztajn,JK(2008)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/18193019”目标=“_ blank”>神经生长因子受体(p75)表达的基础前脑胆碱能神经元的纯化和培养 Nat Protoc 3(1):34-40。
  4. Xu,W.,Weissmiller,AM,White,JA,2nd,Fang,F.,Wang,X.,Wu,Y.,Pearn,ML,Zhao,X.,Sawa,M.,Chen,S.,Gunawardena ,S.,Ding,J.,Mobley,WC and Wu,C。(2016)。  淀粉样蛋白前体蛋白介导的内吞途径破坏引起轴突功能障碍和神经退行性疾病。 J Clin Invest 126(5):1815-1833。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xu, W. and Wu, C. (2017). Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs). Bio-protocol 7(14): e2413. DOI: 10.21769/BioProtoc.2413.
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