小鸡西里神经神经元的分离与培养
Summary
小鸡丝神经通（CG）是寄生虫神经系统的一部分。在神经肌肉相互作用研究中，小鸡CG神经元的神经元培养被证明是有效的细胞模型。我们描述了从小鸡胚胎中解剖、分离和体外培养的详细方案。
Abstract
小鸡丝骨神经合体（CG）是寄生虫神经系统的一部分，负责眼睛中肌肉组织的内膜。这种结节是由一个同质的胆小和胆状神经元组成的，分别内维条纹和平滑的肌肉纤维。这些神经元类型都调节特定的眼部结构和功能。多年来，在肌肉-神经系统相互作用研究中，小鸡胆碱神经的神经元培养被证明是有效的细胞模型，通过胆碱性突触进行交流。胆结神经元，在其大多数，胆碱性。与以前使用的异质细胞模型相比，这种细胞模型已被证明是有用的，这些异构细胞模型包括多种神经元类型，除了胆碱。解剖学上，胆碱结节在视神经（ON）和胆状肌瘤（CF）之间局部。在这里，我们描述了从小鸡胚胎中解剖、分离和体外培养的一个详细程序。我们提供一个分步协议，以获得CG神经元高度纯净稳定的细胞培养，突出过程的关键步骤。这些文化可以在体外保持15天，在此，我们显示了CG文化的正常发展。研究结果还表明，这些神经元可以通过神经肌肉胆碱性突触与肌肉纤维相互作用。
Introduction
Ciliary结节（CG）神经元属于寄生神经系统。这些神经元是胆碱，能够建立肌肉或烟酸突触1，2，3。,31,从解剖学上讲，CG位于视神经（ON）和胆虫肌瘤（CF）之间的眼后部，由大约6000个处于早期胚胎阶段1、4,的神经元组成。在培养的第一周，硅突神经元呈现多极形态。一周后，他们开始过渡到单极状态，一个神经石延伸并形成轴孔5。此外，大约一半的CG神经元死亡之间的8天至14日小鸡胚胎发育，通过细胞死亡的编程过程。神经元数量的减少导致大约3000个神经元6，7，86,的西里结子的总种群。在体外，当生长与肌肉细胞9和CG神经元可以培养几个星期1，9时，CG神经元的数量没有减少。
胆结由同性细胞神经元和胆状神经元的均匀种群组成，每个细胞群代表CG中神经元群的一半，内维眼睛的肌肉。这两种类型的神经元在结构上、解剖学和功能上是截然不同的。虹膜和透镜上的心肌神经元内侧，负责瞳孔收缩。胆道神经元内侧,增强胆道1、10、11、121,10,11的平滑肌。
鸡骨结神经元的培养已被证明是研究神经肌肉突触和突触形成1，5，95的有用工具。1考虑到神经肌肉突触是胆碱13，使用神经群是胆碱性-CG神经元-出现作为一个潜在的替代以前的细胞模型14。这些模型包括异源神经元群体，其中只有一小部分是胆碱性。或者，在体外发育得相对较快，大约15小时后已经形成突触1。CG神经元多年来一直被用作模型系统，用于不同的研究，因为它相对容易分离和操作。这些应用包括光遗传学研究，突触发育，凋亡和神经肌肉相互作用14，15。14,
我们描述了从胚胎第7天（E7）小鸡胚胎中解剖、分离和体外培养的一个详细程序。我们提供一个分步协议，以获得高度纯净和稳定的细胞培养的胆碱神经元。我们还强调了需要特别注意并改善神经元培养质量的协议的关键步骤。这些培养物可以在体外保持至少15天。
Protocol
Representative Results
Discussion
在该协议中，我们演示了如何准备和培养CG神经元。对于没有经验的用户来说，对西里结子的识别和解剖可能很困难。因此，我们提出了一个详细的和分步的程序，以有效地解剖E7小鸡丝骨神经，分离组织，并准备神经元培养，可以保持至少15天。通过此协议获得的 ciliion 节神经元也适合与肌肉细胞共培养。
根据研究的目的，在小鸡胚胎发育的不同发育阶段，可用作细胞模型?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由欧洲区域发展基金（ERDF）通过 CentRO-01-0145-FEDER-00008 项目下的区域业务方案供资：脑健康 2020，CENTRO2020 CENTRO-01-0145-FEDER-00003：pAGE， CENTRO-01-0246-FEDER-00018：MEDISIS，并通过竞争2020 – 竞争力和国际化业务方案，葡萄牙国家基金通过FCT – 基金会，即国际电信协会，在UIDB/04539/2020项目下， UIDB/04501/2020，POCI-01-0145-FEDER-02212：PPBI，PTDC/SAU-NEU/104100/2008，以及个人赠款SFRH/BD/141092/141092/2 2018（M.D.），DL57/2016/CP1448/CT0009（R.O.C.），SFRH/BD/77789/2011（J.R.P.）和玛丽居里行动 – IRG，第七框架方案。
Materials
| 5-fluoro-2’-deoxiuridina (5'-FDU) | Merck (Sigma Aldrich) | F0503 | |
| Alexa Fluor 568-conjugated goat anti-chicken antibody | Thermo Fisher Scientific | A11041 | |
| Alexa Fluor 568-conjugated goat anti-mouse antibody | Thermo Fisher Scientific | A11031 | |
| Alexa Fluor 647-conjugated goat anti-mouse antibody | Thermo Fisher Scientific | A21235 | |
| B27 supplement (50x), serum free | Invitrogen (Gibco) | 17504-044 | |
| Chicken monoclonal neurofilament M | Merck (Sigma Aldrich) | AB5735 | |
| D-(+)-Glucose monohydrate | VWR | 24371.297 | |
| Fetal Bovine Serum (FBS), qualified, Brazil | Invitrogen (Gibco) | 10270-106 | |
| HEPES, fine white crystals, for molecular biology | Fisher Scientific | 10397023 | |
| Horse Serum, heat inactivated, New Zealand origin | Invitrogen (Gibco) | 26050-070 | |
| L-Glutamine (200 mM) | Invitrogen (Gibco) | 25030-081 | |
| Mouse laminin I | Cultrex (R&D systems) | 3400-010-02 | |
| Mouse monoclonal b-III tubulin | Merck (Sigma Aldrich) | T8578 | |
| Mouse monoclonal SV2 | DSHB | AB2315387 | |
| Multidishes, cell culture treated, BioLite, MW24 (50x) | Thermo Fisher Scientific | 11874235 | |
| Neurobasal medium without glutamine | Invitrogen (Gibco) | 21103-049 | |
| Penicillin/streptomycin (5,000 U/mL) | Invitrogen (Gibco) | 15070-063 | |
| Phenol red, bioreagent, suitable for cell culture | Merck (Sigma Aldrich) | P3532 | |
| Poly-D-Lysine | Merck (Sigma Aldrich) | P7886 | |
| Potassium chloride | Fluka (Honeywell Reaarch Chemicals) | 31248-1KG | |
| Potassium di-hydrogen phosphate (KH2PO4) for analysis, ACS | Panreac Applichem | 131509-1000 | |
| Prolong Gold Antifade mounting medium with DAPI | Invitrogen (Gibco) | P36935 | |
| Puradisc FP 30mm Syringe Filter, Cellulose Acetate, 0.2µm, sterile 50/pk | Fisher Scientific | 10462200 | |
| Recombinant human ciliary neurotrophic factor (CNTF) | Peprotech | 450-13 | |
| Recombinant human glial cell-derived neurotrophic factor (GDNF) | Peprotech | 450-10 | |
| Sodium chloride for analysis, ACS, ISO | Panreac Applichem | 131659-1000 | |
| Sodium dihydrogen phosphate 2-hydrate (Na2HPO4·2H2O), pure, pharma grade | Panreac Applichem | 141677-1000 | |
| Sodium Pyruvate 100 mM (100x) | Thermo Fisher | 11360039 | |
| Syringe without needle, 10 mL | Thermo Fisher | 11587292 | |
| Trypsin 1:250 powder | Invitrogen (Gibco) | 27250-018 |
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