Name: levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions
Uploaded: 2018-05-05T14:00:00
Description: Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

我们感谢马克 m. 富先生和丹尼尔. 米兰达的社论评论, 艾哈迈德. Khedraki 帮助建立了这项技术, 莱特州立大学提供财政支持 (启动基金到 A.A.V.)。

所有动物程序都是按照赖特州立大学动物保育和使用委员会的规定进行的。
1. 老鼠安乐死
<ol><li>在通风罩中, 将鼠标放在密闭玻璃麻醉室中。</li>
	<li>将老鼠吸入到致命剂量的异氟醚 (饱和, 或 ~ 25%)。把老鼠留在房间里, 直到呼吸不到为止。</li>
	<li>将小鼠从腔室取出, 并作为安乐死的次要方法进行颈椎脱位。</li>
</ol>2. 从头部、颈部和背部的背部表面取出毛发...

<ol>
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这里描述的是制备和使用的小鼠拉尔肌肉测量神经肌肉传输在电流或电压钳条件下。解剖拉尔有几个重要的要点要考虑。在电极掷镖师掷中清除肌肉辅助组织中多余的结缔组织, 因为电极在定位掷镖师掷时可能会感染结缔组织。然而, 只有去除结缔组织, 可以很容易地被带走, 以限制伤害的机会, 肌肉。神经的隔离应该小心地进行, 因为它非常微妙。为了避免神经损伤, 将一些周围的组织连接到神经?...

研究神经肌肉连接处的突触传递, 可以洞察神经和骨骼肌肉系统之间的动态关系, 是检查突触生理学的绝佳模型。提肛耳肌 (拉尔) 是一种薄肌肉, 允许神经肌肉连接容易形象化。以前的报告描述了使用拉尔检查突触药物和毒素的便利性, 并描绘了拉尔12的骨骼肌纤维类型特征。许多研究使用了拉尔检查神经肌肉生理学3,4,5,6,7,8。对于电生理, 能够轻松地观察拉尔神经肌肉连接允许准确地放置电极在电机终板, 并大大减少空间钳位问题记录突触传输。现有的肌肉膜性能的钳位记录, 如膜时间常数 (τm) 和输入电阻 (R in) 是容易获得的.此外, 这些特性可以用用于记录神经肌肉传输的相同肌肉纤维来测量, 从而直接将突触功能与肌肉膜的特性进行比较。对这些数据的分析可以为许多神经肌肉疾病的物理机制和改变活动状态提供关键的洞察力。
这里描述的技术的一个关键方面是使用电压钳的突触录音, 这是不受非非线性遇到的电流钳和独立的肌肉膜的性质。使用电压钳, 而不是电流钳检查神经肌肉传输的优点是通过二十世纪五十年代9的开创性努力建立的。在电流钳下, 在振幅超过 10-15 mV 的无害产品不是 mEPP 振幅9的线性乘积。例如, 如果平均 mEPP 是 1 mv, 5 mv 的 EPP 可以假设是 5 mEPPs 的产品 (QC 5);然而, 40 mV 的 EPP 将是超过 40 mEPPs 的产品。这种非线性在更大的无害性发生, 因为 EPP 的驱动力, 这是不同的膜电位和平衡电位的乙酰胆碱受体 (10 mV), 大大减少在大型无害性。在电压钳实验中, 由于肌肉膜电位没有改变, 所以在压夹试验中避免了这个问题。缺点是, 在技术上比电流钳记录更难完成电压钳的实验。考虑到这一点, McLachlan 和马丁开发了一个简单的数学校正, 它在非线性10的当前钳型录音中占了不可用的。更正工作良好11,12,13, 但重要的是, 假设肌肉膜的性能没有中断。
肌肉膜的性质是特别重要的考虑, 如果研究条件或疾病状态, 扰乱肌肉。例如, R6/2 基因模型的骨骼肌从亨廷顿氏病是 hyperexcitable 由于逐步减少休息氯和钾电流14,15。结果, mEPPs 和无害的 R6/2 骨骼肌肉被放大。当然, 其他因素可以改变 mEPPs 和无害环境。与亨廷顿病小鼠 (R6/1) 的不同模型工作发现了无害性的变化, 似乎与诱捕蛋白8有关。为了评估导致神经肌肉传导改变的机制, 用电压钳消除改变肌肉膜性能的效果将是有益的。在最近的一项研究中, R6/2 神经肌肉传输的研究在电流和电压钳条件下使用的技术描述。通过将两个电极放在板板16的长度常数内, 电机终板的整体被压紧, 误差小于1%。结果表明, 电压钳和校正的电流钳记录产生了 R6/2 肌神经肌肉传递的对比测量。这突出表明, 如果肌肉膜的性能已经改变, 并显示获得独立于肌肉膜性能的电压钳记录的好处, 则可能很难纠正非非线性的无害化。本文提出的协议是检查影响突触传递和突触后膜性能的条件或疾病状态的理想选择。

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus Compound Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">UMPLFLN10XW</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">LUMPLFLN40XW</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:216px;">Borosilicate Glass</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">BF150-86-7.5</td>
			<td style="width:288px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CCD Camera</td>
			<td style="width:183px;">Santa Barbara Instruments Group</td>
			<td style="width:119px;">ST-7XMEI</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:216px;">Axoclamp 900A Amplifier</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;">2500&#8208;0179&#160;&#160;</td>
			<td style="width:288px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mater-9 Pulse Generator</td>
			<td style="width:183px;">AMPI</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;width:216px;">Iso-flex Stimulus Isolator</td>
			<td style="width:183px;">AMPI</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pCLAMP 10 Data Acquisition and Analysis Software</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;">1-2500-0180</td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;width:216px;">Concentric Bipolar Electrode</td>
			<td style="width:183px;">FHC</td>
			<td style="width:119px;">CBDSH75</td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;width:216px;">Ball-joint Manipulator</td>
			<td style="width:183px;">Narishige&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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			<td height="52" style="height:52px;width:216px;">Non-metalic Syringes 34 Gauge</td>
			<td style="width:183px;">World Precision Instruments</td>
			<td style="width:119px;">MF34G-5</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nikon Stereomicroscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 5 Forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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			<td height="26" style="height:27px;width:216px;">Spring Scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;width:216px;">No. 2 Forceps</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5Q41</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microdissecting Scissors</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5912SC</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:183px;">Dow Corning</td>
			<td align="right" style="width:119px;">2404019862</td>
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			<td height="21" style="height:21px;width:216px;">Hair Removal Cream</td>
			<td style="width:183px;">Nair</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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		<tr height="27">
			<td height="27" style="height:27px;width:216px;">Grass SD9 Stimulator</td>
			<td style="width:183px;">Grass Medical</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Model P-1000 Micropipette Puller</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Axon Digidata 1550 Low-noise Data Acuisition System</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Low Pass Bessell Filter</td>
			<td style="width:183px;">Warner Instrument Corp.</td>
			<td style="width:119px;">LPF-8</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Left-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DL</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Right-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DR</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Single Motion Controler</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MC100e</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:216px;">Crossed Roller Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641R</td>
			<td style="width:288px;">This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:517px;">All chemicals were orded from Fisher except,</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTS</td>
			<td style="width:183px;">Toronto Research Chemicals</td>
			<td style="width:119px;">B315190</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CTX</td>
			<td style="width:183px;">Alomone Labs</td>
			<td style="width:119px;">C-270</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-Di-2-Asp</td>
			<td style="width:183px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:288px;">Molecular probes is no longer a company. Now ordered through Fisher</td>
		</tr>
	</tbody>
</table>

levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions

本协议描述了一种在电流钳和电压钳条件下记录神经肌肉结突触传递的技术。使用肛提耳肌 (拉尔) 的前体制备, 是因为它是一种薄肌肉, 可在马达终板上为微电极掷镖师掷提供易于可视化的神经肌肉连接。该方法允许记录自发微型终板电位和电流 (mEPPs 和 mEPCs), 神经诱发终板电位和电流 (无害性和 EPCs), 以及电机终板的膜性能。从该方法得到的结果包括量子含量 (QC), 囊泡释放部位的数量 (n), 囊泡释放的可能性 (p相对), 突触促进和抑郁症, 以及肌肉膜时间常数 (τm) 和输入电阻。这种技术应用于人类疾病的小鼠模型可以突出疾病状态中的关键病理, 并有助于确定新的治疗策略。通过完全压紧单个突触, 该方法提供了目前可用的突触传输的最详细的分析之一。

图 8显示了当前的脉冲 (图 8A) 和电压响应 (图 8B) 的一个例子, 从一个拉尔纤维下的电流钳, 从一个12周大的野生类型 R6/2 鼠标。mEPPs 的存在表明这些记录是从马达终板中取出的。这些记录是在正常的生理盐水溶液中获得的。可以分析这些当前钳位记录, 以确定该光纤16、<sup...

Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

本文所描述的协议使用鼠标提耳肌 (拉尔) 肌肉记录自发性和神经诱发突触后电位 (电流钳) 和电流 (电压钳) 在神经肌肉连接。使用该技术可以为正常和疾病条件下突触传递机制提供关键的见解。

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Research

JoVE Journal

Neuroscience

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Measuring Neuromuscular Junction Functionality

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...