Name: making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain
Uploaded: 2018-05-07T14:45:00
Description: Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

Khakh 实验室得到了 NIH MH104069 的支持。这个实验室是由 NIH NS030549 支持的。J.C.O. 感谢 NIH T32 神经集成电路训练补助金 (NS058280)。

所有动物实验都是按照美国国家卫生研究院的实验室动物护理和使用指南进行的, 并得到了加州大学洛杉矶分校校长动物研究委员会的批准。所有的老鼠都是在12小时的昏暗环境中使用食物和水的ad 随意。所有动物都是健康的, 没有明显的行为变化, 没有参与以前的研究, 并在光周期中牺牲。实验数据从成年小鼠 (6-8 周大的所有实验) 中采集。
1. K+选择性电极的制备...

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我们在这里描述的方法使我们能够评估 K+动力学, 以响应电刺激的谢弗络在急性海马切片的成年小鼠。我们准备 K+离子选择性电极的方法类似于前面描述的过程12,13,14,15。然而, 这种方法优于替代电极配置, 因为它是快速和复杂的准备 K+选择性电极。在适当的校准后, 发现这些电...

钾离子浓度在大脑中受到严格的调节, 它们的波动对所有细胞的静止膜电位有很大的影响。根据这些重要的贡献, 生物学的一个重要目标是确定细胞和生物物理机制, 用于在身体的不同器官的细胞外空间中严格调节 K+的浓度1,2. 这些研究的一个重要要求是能够准确地测量 K+浓度。虽然在健康和患病状态下, 许多有助于大脑中钾稳态的成分已经被确认为3,45, 由于专业性质的原因, 进一步的进展已经减慢。制备离子选择性电极钾的方法。微电极传感器表示用于测量 K+浓度体外、组织切片和体内中的金标准。
用于 k+监视的更新方法正在使用光学传感器进行开发, 但这些检测不到与生物学相关的 k+浓度范围, 也未在生物系统中进行全面审查, 尽管初步结果显示有希望的6,7,8。与光学传感器相比, 电极从根本上被限制为离子的点源测量, 虽然电极阵列可以改善空间分辨率9。本文重点介绍了用于监测 K+动力学的单桶微电极传感器。
在这项工作中, 我们报告详细的逐步过程, 使 K+选择性电极, 使用基于 valinomycin 的钾载体, 允许高度选择性 (104折叠 k+到 Na+选择性) k+在膜上运动10。valinomycin 是一种自然发生的多肽, 它充当 k+渗透性孔隙, 并使 k+的流量降低到其电化学梯度。我们还描述了如何校准电极, 如何存储和使用它们, 最后如何部署它们来测量成年小鼠海马脑切片中的 K+浓度动态。使用此类电极和基因修饰小鼠缺乏特定的离子通道来调节胞外 K+动力学应该揭示神经系统使用的细胞机制来控制 k 的环境浓度。在胞外环境中的+ 。

<table border="0" cellpadding="0" cellspacing="0" width="997">
	<tbody>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Vibratome</td>
			<td style="width:241px;">DSK</td>
			<td style="width:257px;">Microslicer Zero 1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Mouse: C57BL/6NTac inbred mice</td>
			<td>Taconic</td>
			<td style="width:257px;">Stock#B6</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Electrode puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Ag/AgCl ground pellet</td>
			<td>WPI</td>
			<td>EP2</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">pCLAMP10.3</td>
			<td style="width:241px;">Molecular Devices</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Custom microfil 28G tip</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">CMF28G</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Tungsten Rod</td>
			<td style="width:241px;">A-M Systems</td>
			<td style="width:257px;">716000</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Bipolar stimulating electrodes</td>
			<td style="width:241px;">FHC</td>
			<td style="width:257px;">MX21XEW(T01)</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Stimulus isolator</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">A365</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Grass S88 Stimulator</td>
			<td style="width:241px;">Grass Instruments Company</td>
			<td style="width:257px;">S88</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Borosilicate glass pipettes</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">1B150-4</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">A to D board</td>
			<td style="width:241px;">Digidata 1322A</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Signal Amplifier</td>
			<td style="width:241px;">Multiclamp 700A or 700B</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Headstage</td>
			<td style="width:241px;">CV-7B Cat 1</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Patch computer</td>
			<td style="width:241px;">Dell</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5886</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">P3911</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">HEPES</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H3375</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Bicarbonate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5761</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Phosphate Monobasic</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S0751</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">D-glucose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">G7528</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Calcium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">21108</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Magnesium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">M8266</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">valinomycin</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">V0627-10mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">1,2-dimethyl-3-nitrobenzene</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">40870-25ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium tetrakis (4-chlorophenyl)borate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">60591-100mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">5% dimethyldichlorosilane in heptane</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">85126-5ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">TTX</td>
			<td style="width:241px;">Cayman Chemical Company</td>
			<td style="width:257px;">14964</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Hydrochloric acid</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H1758-500mL</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sucrose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S9378-5kg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Pipette Micromanipulator</td>
			<td style="width:241px;">Sutter</td>
			<td style="width:257px;">MP-285 / ROE-200 / MPC-200</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Objective lens</td>
			<td style="width:241px;">Olympus</td>
			<td style="width:257px;">PlanAPO 10xW</td>
			<td></td>
		</tr>
	</tbody>
</table>

making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain

钾离子对细胞的静止膜电位有显著的促进作用, 因此胞外 K+浓度是细胞兴奋性的重要调节因子。细胞外 K+的浓度改变会影响静止膜电位和细胞兴奋性, 方法是将闭合的、开放的和灭活状态之间的平衡转移到有作用电位的电压依赖离子通道上。启动和传导。因此, 直接测量健康和患病状态下的胞外 K+动态是很有价值的。在这里, 我们描述如何制作, 校准和使用单 K+选择性电极。我们将它们部署在成人海马脑切片中, 以测量电诱发 K+浓度动态。明智地使用此类电极是评估细胞和生物物理机制, 以控制神经系统细胞外 K+浓度所需的工具套件的重要组成部分。

对于细胞外 K+的选择性测量, 我们通过硅烷化清洁硼硅酸盐玻璃吸管(图 1A), 制备了具有疏水性层的离子选择性电极。此涂层使包含 valinomycin 的 k+载体在电极尖端上休息, 并且仅允许 k+通量通过在电极尖端的窄开口 (图 1B)。在用回填盐水溶液和 K+载体启动电极后, 可以对电极进行测试, 使其快速、?...

Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

成人脑组织切片中钾离子选择性电极的制备、检测及应用

钾离子有助于细胞的静止膜电位, 胞外 K+浓度是细胞兴奋性的重要调节因子。我们描述如何制作, 校准和使用单 K+选择性电极。使用此类电极可以测量成年海马切片中电诱发的 K+浓度动态。

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Research

JoVE Journal

Neuroscience

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

科研

神经科学

Subtype-selective Electroporation of Cortical Interneurons

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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 ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

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 ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

使用基于 FRET 的传感器 ATeam1.03YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...