Name: healthy brain-pituitary slices for electrophysiological investigations of pituitary cells in teleost fish
Uploaded: 2018-08-16T12:30:00
Description: Watch this Scientific Journal Video about Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish at JoVE.com

我们感谢 LourdesCarreon 女士帮助维护鳉设施和安东尼. 珀尔为例证数字。这项工作由 NMBU 和挪威研究理事会资助, 赠款244461号 (水产养殖项目) 和 248828 (数字生活挪威计划)。

所有动物处理都是根据挪威生命科学大学研究动物的护理和福利的建议进行的, 并在授权调查员的监督下进行。
1. 编写文书和解决办法
注意: 所有的解决方案都应该是无菌的。应认真注意所有溶液的 pH 值和渗透压 (osmol/千克水), 应仔细地适应所研究物种的胞外环境。ph 值和渗透率应与精密电子设备 (如 ph 表和冰点 osmometer) 进行调整。
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用膜片钳技术进行脑-垂体切片的电生理记录需要仔细的优化。在硬骨鱼类中专门进行活细胞调查的优化协议是有限的, 大多数出版物都使用基于哺乳动物系统的协议。在这方面, 重要的是要知道, 一些生理参数, 如 pH 值和渗透系数不仅是物种依赖, 而且很大程度上取决于有关有机体是否生活在陆地上或在水中。对鱼类而言, 如果它们生活在海洋或淡水环境中, 也必须考虑到这一点。例如, 在鱼类中, CO...

垂体是脊椎动物的主要内分泌器官, 位于下丘脑下和视神经交叉后。它产生和分泌六到八荷尔蒙从特定细胞类型。垂体激素是大脑和周围器官之间的一种中间体, 它推动了一系列重要的生理过程, 包括生长、繁殖和调节稳态。与神经元相似, 垂体内分泌细胞具有自发激发动作电位1的电兴奋能力。这些动作电位的作用是细胞依赖。在哺乳动物垂体的几种细胞类型中, 动作电位可以使细胞内钙含量提高2 以上, 以持续释放荷尔蒙2。此外, 垂体从大脑中得到刺激和抑制信息, 影响细胞3、4、5、6的细胞膜电位。通常情况下, 刺激输入会增加兴奋性, 并且经常涉及从胞内存储中释放 Ca2 +以及增加射击频率7。了解细胞如何利用离子通道组成, 并适应这些输入信号从大脑是了解激素合成和释放的关键。
二十世纪七十年代下旬, Sakmann 和内尔8、9、10和哈米尔11进一步改进了膜片钳技术, 并允许对细胞的电生理特性进行详细的研究。单离子通道。此外, 该技术还可用于研究电流和电压。今天, 贴片夹紧是衡量细胞电生理特性的金标准。四密密封贴片钳技术主要配置11;细胞连接, 内而外, 外出, 和整个细胞补丁。三第一种配置通常用于单离子通道调查。第四, 在细胞连接配置后, 细胞膜上的一个孔是利用亚大气压进行的。这种配置还允许对整个单元12的离子通道组成进行调查。然而, 这种技术的一个局限性是, 细胞质分子是稀释的膜片吸管溶液13 (图 1A), 从而影响的电学和生理反应的研究细胞。事实上, 其中一些分子可能在信号传导或不同离子通道的调节中扮演重要角色。为了避免这种情况, 林道和费尔南德斯14开发了一种方法, 将孔隙形成的化合物添加到贴片吸管中。根据细胞连接的配置, 该化合物将并入等离子膜下的补丁和慢慢穿孔的膜创建电接触细胞质 (图 1B)。几种不同的抗真菌药物, 如制霉菌素15和两性霉素 B 16, 或表面活性剂, 如皂苷β-七叶皂素17,18可以使用。这些化合物创造足够大的毛孔, 允许在细胞质和贴剂之间进行单价阳离子和 Cl 扩散, 同时保持大分子和更大的离子如 Ca2 + 15的胞浆水平, 16。
使用穿孔补丁的挑战是潜在的高串联电阻。串联电阻 (Rs) 或接触电阻是相对于地面的贴片吸管的联合电阻。在膜片钳录音中, rs将与膜电阻 (rm)平行。rm和 rs并行工作, 作为一个电压分配器。随着高 rs,电压将下降的 rs给错误的录音。随着更大的电流记录, 错误将变得更大。此外, 分压器还依赖于频率依赖性的创建低通滤波器, 从而影响时间分辨率。实际上, 穿孔补丁可能并不总是允许大和快速电流的记录, 如电压门控 Na+电流 (详细读数见参考19)。另外, Rs在补丁钳录音期间可能变化, 再导致记录的当前的变动。因此, 在药物应用过程中 R 变化的情况下, 可能发生误报。
安徒生实验室首先介绍了切片组织的电生理学, 以研究脑20中神经元的电生理特性。该技术为在更完整的环境中对单细胞以及细胞通讯和细胞电路的详细研究铺平了道路。1998年 Guérineau 了一种类似的制作垂体切片的技术. 21. 然而, 2005年之前, 脑-垂体切片制剂已成功用于硬骨鱼类22的膜片钳研究。在本研究中, 作者还报告了使用穿孔膜片钳录音。然而, 到目前为止, 大多数垂体细胞的电生理研究都是在哺乳动物中进行的, 只有少数其他脊椎动物, 包括硬骨鱼类鱼1,2,22,23.在硬骨鱼类, 几乎所有的研究都在主要的离解细胞24,25,26,27,28,29,30 进行.
本文概述了从模型鱼鳉中制备健康脑-垂体切片的优化方案。与原代分离细胞培养相比, 该方法具有多种优点。首先, 与分离细胞培养条件相比, 细胞在相对保存的环境中被记录下来。第二, 切片制剂使我们能够研究细胞通讯介导的间接通路22, 这在分离细胞培养条件下是不可能的。此外, 我们还演示了如何使用多孔全细胞膜片钳技术, 以两性霉素 B 为成孔剂, 对获得的组织切片进行电生理记录。
鳉是原产于亚洲的一条小型淡水鱼, 主要见于日本。鳉的生理、胚胎学和遗传学研究已有100年的31, 是许多实验室中常用的研究模式。本文特别重视硬骨鱼类鱼下丘脑-垂体复合体的形态学组织: 而在哺乳动物和鸟类中, 下丘脑神经元释放其调节垂体内分泌细胞的神经激素。进入中位隆起的门系统, 有一个直接的神经投射到下丘脑神经元的内分泌细胞的垂体在硬骨鱼类鱼32。因此, 仔细进行脑-垂体切片在鱼类中尤为重要, 使我们能够研究保存完好的脑-垂体网络中垂体细胞的电生理特性, 特别是垂体细胞如何控制他们的兴奋性, 从而 Ca2 +稳态。

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healthy brain-pituitary slices for electrophysiological investigations of pituitary cells in teleost fish

垂体细胞的电生理研究已经在许多脊椎动物中进行, 但在硬骨鱼类鱼中很少。其中, 明显多数是在分离的主要细胞上进行的。为了提高我们对如何硬骨鱼类垂体细胞的理解, 在一个更具生物学意义的环境中表现, 这个协议显示了如何使用小淡水鱼鳉 (Oryzias latipes) 制备可行的脑垂体切片。将所有溶液的脑-垂体切片、pH 值和渗透压调整到生活在25到28摄氏度的淡水鱼体液中。在切片准备后, 该协议演示了如何使用穿孔的全细胞膜片钳技术进行电生理记录。膜片钳技术是一种功能强大的工具, 具有空前的时间分辨率和灵敏度, 允许从完整的整个细胞到单个离子通道的电性能进行调查。穿孔补丁是独一无二的, 因为它保持细胞内环境的完整性, 防止调节元素的细胞质被稀释的补丁吸管电极解决方案。相比之下, 在执行传统的全细胞记录时, 观察到鳉垂体细胞很快就失去了射击动作电位的能力。在现有的各种穿孔技术中, 本协议演示了如何使用杀菌剂两性霉素 B 实现修补膜穿孔。

该协议演示了如何通过鳉转基因线 [Tg (lhb-hrGfpII)] 在目标细胞 (Lh 产生的 gonadotropes) 中实现可靠的电生理记录, 从垂体 (gonadotrope) 细胞中获得可靠性的一步一步的协议。被标记为绿色荧光蛋白 (GFP)。
初步采用全细胞构型进行电生理研究。然而, 任何研究的细胞都没有观察到自发动作电位。在细胞的子集中, 动作电位可以通?...

Watch this Scientific Journal Video about Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish at JoVE.com

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

本文介绍了利用硬骨鱼类鱼鳉 (Oryzias latipes) 制作可行的脑-垂体组织切片的优化协议, 然后用膜片钳技术进行垂体细胞的电生理记录, 并穿孔补丁配置。

健康脑-垂体切片对硬骨鱼类鱼垂体细胞电生理的研究

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

Research

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Neuroscience

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

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神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

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Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

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在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

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在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

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视网膜原酸和皮质化突触功能的电生理学研究

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...