通过特洛多毒素微注射可逆灭活，揭开鼠脑离散区在排卵调节中的作用
1Chronobiology of Reproduction Research Lab, Biology of Reproduction Research Unit, Facultad de Estudios Superiores Zaragoza,Universidad Nacional Autónoma de México, 2Developmental Biology Lab, Biology of Reproduction Research Unit, Facultad de Estudios Superiores Zaragoza,Universidad Nacional Autónoma de México, 3Neuroendocrinology Lab, Biology of Reproduction Research Unit, Facultad de Estudios Superiores Zaragoza,Universidad Nacional Autónoma de México, 4Facultad de Ciencias,Universidad Nacional Autónoma de México, 5Department of Basic Research, Instituto Nacional de Geriatria,Ministry of Health
Summary
本协议描述了低成本微注射系统的构建、对深脑结构的立体定向植入以及清醒和无拘无束的大鼠对四毒素定时微注射的程序。目标是通过抑制下丘脑结构的神经活动来揭示下丘脑结构参与排卵的调节。
Abstract
许多实验方法被用于研究大脑在排卵调节中的作用。例子包括神经元组的病变和耳聋，这两种侵入性方法都永久地损害了目标区域的完整性。这些方法还附带附带影响，可影响对急性和时效监管机制的分析。针对特定大脑区域的导引罐体的立体植入，然后是恢复期，使研究人员能够在手术的不良效果消失后微观注射不同的药物。四毒素已被用于确定几个大脑区域在各种生理过程中的作用，因为它暂时抑制了依赖钠的作用潜力，从而阻断了目标区域的所有神经活动。本协议将此方法与评估卵巢周期和排卵的策略相结合，以揭示离散的大脑区域在排卵调节中在最恶劣周期的任何特定阶段的作用。觉醒和无拘无束的老鼠（Rattus norvegicus）被用来避免麻醉剂和应激激素对排卵的阻塞作用。此协议可以很容易地适应其他物种，大脑目标和药理制剂，以研究不同的生理过程。该方法的未来改进包括使用小直径的玻璃毛细血管而不是导引罐体设计微型喷射系统。这将减少植入过程中受损的组织数量，并减少注入药物在目标区域外的扩散。
Introduction
排卵是每个卵巢/月经周期从卵巢中释放一个或多个成熟卵母细胞的过程。由于所有哺乳动物物种都依赖于游戏品种的生产，对调节排卵机制的理解在生物医学、畜牧业和濒危物种的维持等领域产生了巨大影响。排卵由下丘脑-垂体-卵巢轴调节，该轴涉及几个下丘脑和下垂外区域、前垂体中的角膜和肉芽细胞，这些细胞与卵母细胞一起形成卵巢内的卵巢卵泡1。
卵巢卵泡生长，发展，并最终排卵，以回应卵泡刺激激素和叶黄素化激素的补品和阶段性分泌，由卵泡激素分泌的两个卵巢激素。戈纳多特罗平分泌模式是适当的卵泡发育和排卵的关键，它是由戈纳多特罗平释放激素 （GnRH）1，2调节。这种神经肽由分散在基底脑中的神经元合成，然后分泌到连接下丘脑和前垂体的门户血管。GnRH神经元的分泌活动反过来又由不同大脑结构产生的突触输入调节。这些结构传达了有关生物体外部和内部环境状况的信息，包括食物的供应、光佩里德的长度和血液中激素的浓度。从这个意义上说，它们塑造了每个物种的生殖模式，必须确定这些结构的具体作用，以便正确理解排卵的机制。例如，已经表明，在最恶劣的周期中，雌二醇水平的波动调节了GnRH的分泌：然而，GnRH神经元并不表示检测此类变化所需的雌二醇受体等形。表达这些受体的两个神经元群分别位于第三心室的椎间垂直区域和弧形细胞核中，并且与GnRH神经元有稳定突触。有证据表明，这些神经元解释雌二醇的浓度，然后刺激GnRH神经元的活性，释放吻肽，GnRH分泌物3的有力诱导剂。
涉及热病变或化学病变以及机械耳聋的实验使研究人员能够确定几个大脑结构参与排卵4、5、6、7、8、9、10、11、12的调节.然而，这些实验具有侵入性和创伤性的缺点，在评估治疗效果之前需要几天的恢复时间，从而妨碍了对治疗急性效应的分析。此外，它们长期影响目标区域并中断其他生理过程。由于这些问题，这些实验的结果通常被动物体内的内在补偿机制所掩盖，并提取有关所涉及区域的时间调节动态的准确信息是相当困难的。
通过引导性大麻酶暂时破坏神经元活动的药物的微注射是一种适当的替代方法，可以超越上述缺点。通过立体定向手术，可以将金核酸放入任何大脑区域，使研究人员能够在手术的混淆效应消失后开始药物治疗。药物的定时微注射使研究人员能够测试有关该地区对过程特定步骤的贡献的假设，并且可以在清醒的受约束或自由移动的动物中执行。各种药物，包括局部麻醉剂、激动剂、拮抗剂、反激动剂和生物毒素，如四毒素（TTX），可在特定时间微注射到感兴趣的区域。
TTX是一种生物毒素，由生活在河豚体内的细菌以及其他脊椎动物和无脊椎动物合成。TTX通过对钠通道的选择性和瞬态封锁来抑制神经活动，从而抑制依赖钠的行为潜力。在TTX的存在下，细胞在去极化阶段经历改变，因此不能激发，但仍然活着。TTX的阻塞效应是由其分子组成解释的：一个硅基组能够通过钠通道的细胞外方面，但分子的其余部分由于其大小而无法通过，因此它被卡住并阻塞了通道13、14、15、16、17.TTX 的作用机制允许它作为研究体外和体内神经系统的工具。这种毒素的内脑注射已用于研究离散脑区在几个过程中的作用，如记忆保留18，睡眠和觉醒19，位置识别20，空间导航21，药物滥用22，热调节23，精神分裂症24的发展，性行为25和排卵调节26 除其他外。在这个协议中，我们描述了在清醒和无拘无束的大鼠中TTX微注射对下丘脑核短暂灭活排卵的影响。
Protocol
涉及动物的程序已得到联阿特派团萨拉戈萨省高级法库塔德道德委员会的批准。该机构严格按照墨西哥动物处理规则（官方规范：NOM-062-ZOO-1999）运作，该规则符合国际准则。 1. 建设双边坎努拉斯 使用压力钳从两根 23 G 皮下针的中心提取不锈钢轴，然后使用手术刀刀片去除剩余的胶水。 绘制一条线 15 毫米，从轴的钝端与精细的永久标记。使用切割钳子去除斜?…
Representative Results
上述协议通过评估单个TTX或车辆（人工脑脊液）的影响进行了测试：ACSF）微注射成两个不同的细胞核之一，已知参与调节排卵在老鼠：超囊性原子核和弧形核。之所以选择超乳酸核，是因为它含有哺乳动物的中央昼夜起搏器。它参与循环事件的监管，作为戈纳多特罗平的分泌物。之所以选择弧形细胞核，是因为它包含大量表达雌二醇受体的神经元，在大多数绝经周期中刺激GnRH分泌。微注射是在14…
Discussion
本文描述了一种在任何给定时间暂时灭活清醒和无拘无束的老鼠大脑中一个离散区域的方法。还提供了一个简单的方法来跟踪他们的排卵周期和评估排卵。此协议允许通过比较TTX处理动物的排卵结果与车辆处理动物的排卵结果，直接分析特定大脑区域对驱动排卵机制的贡献。除了神经科学实验室常见的立体定向仪器和微注射泵外，这种方法不需要昂贵的材料。商用罐体和微喷射器是这类实验的常?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢华盛顿大学的雷蒙德·桑切斯在手稿编辑和 M.Sc 方面给予的宝贵帮助。 乔治娜·科尔特斯和辛蒂亚·哈维尔 M.Sc 在这项技术标准化方面提供了技术支持。我们也感谢萨拉戈萨省高级兽医服务机构的成员：MVZ。阿德里亚娜·阿尔塔米拉诺，MVZ罗曼·埃尔南德斯和MVZ多洛雷斯-伊丽莎白·古斯芒为实验动物提供出色的维护和护理。本协议中描述的实验得到了DGAPA-PAPIIT赠款编号：IN216015和CONACyT赠款编号的支持：236908罗伯托·多明格斯。卡洛斯-卡米洛·席尔瓦是墨西哥国立大学生物学博士项目的博士生，并得到墨西哥国家科学与技术委员会（赠款编号：294555）的支持。
Materials
| 10 μL Hamilton syringes | Hamilton | 80314 | |
| 21 G x 1" stainless steel hypdermic needle | BD | 305165 | |
| 23 G x 1" stainless steel hypdermic needle | BD | 305145 | |
| 30 G x 1/2" stainless steel hypdermic needle | BD | 305106 | |
| Artificial cerebrospinal fluid | BASi | MD-2400 | |
| Bone trimer | Fine Science Tools | 16152-12 | |
| Burr for micro drill | Fine Science Tools | 19007-05 | |
| Clipper | Wahl | ||
| Cut-off disc | Dremel | SM5010 | |
| Cutting tweezers | Truper | 17367 | |
| Cyanocrylate glue | Kola loka | K-1 | |
| Dental cement | Nic Tone | ||
| Enrofloxasin | Senosiain | ||
| Eosin | Sigma | E4009 | |
| Estereoscope | Zeiss | ||
| Extra fine Bonn scissors | Fine Science Tools | 14084-08 | |
| Face mask | Lanceta HG | 60036 | |
| Graefe Forceps | Fine Science Tools | 11050-10 | |
| Hematoxilin | Sigma | H3136 | |
| Hemostats | Fine Science Tools | 13008-12 | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | |
| Hydrochloric acid | Sigma | 320331 | |
| Hypromelose artificial tears | Sophia Labs | 8950015 | |
| Isoflurane | Pisa Agropecuaria | ||
| Meloxicam | Aranda | 1183 | |
| Microinjection pump | KD Scientific | 788380 | |
| Monomer | Nic Tone | ||
| Mototool | Dremel | 3000 | |
| Nitrile gloves | Lanceta HG | 69028 | |
| Non-Rupture Ear Bars | David Kopf Instruments | 855 | |
| Poly-L lysine | Sigma | P4707 | |
| Povidone-iodine | Dermo Dine | ||
| Povidone-iodine with soap | Germisin espuma | ||
| Pressure tweezers | Truper | 17371 | |
| Rat anesthesia mask | David Kopf Instruments | Model 906 | |
| Saline solution | PISA | ||
| Scalpel | Fine Science Tools | 10004-13 | |
| Scalpel blade | Fine Science Tools | 10015-00 | |
| Sodium pentobarbital | Pisa Agropecuaria | ||
| Standard electrode holder | David Kopf Instruments | 1770 | |
| Stainless steel wire | American Orthodontic | 856-612 | |
| Stereotaxic apparatus | David Kopf Instruments | Model 900LS | |
| Surgical Sissors | Fine Science Tools | 14001-12 | |
| Teflon connectors | Basi | MD-1510 | |
| Teflon tubing | Basi | MF-5164 | |
| Tetrodotoxin | Alomone labs | T-500 | |
| Vaporizer | Kent scientific | VetFlo |
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Silva, C., Bolaños-Hurtado, M., Juárez-Tapia, C., Flores, A., Arrieta-Cruz, I., Cruz, M., Domínguez, R. Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections. J. Vis. Exp. (163), e61493, doi:10.3791/61493 (2020).