Name: desensitization and recovery of crayfish photoreceptors upon delivery of a light stimulus
Uploaded: 2019-11-09T12:00:00
Description: Watch this Scientific Journal Video about Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus at JoVE.com

这项工作得到了DGAPA-UNAMIN224616-RN224616赠款的支持。作者要感谢联合国妇女署医学学院科学论文翻译部主任何塞菲娜·博拉多夫人编辑本手稿的英文版本。

注：实验符合墨西哥动物保护法。
1. 实验设置
<ol><li>常规连接<ol>
<li>通过模拟到数字转换器将放大器连接到合适的计算机，并使用示波器监控实验（图1）。</li>
		<li>将照片模拟器连接到转换的 A/D。</li>
	</ol>
</li>
	<li>录音室<ol>
<li>将记录室放在防振台的顶部，并将其放置在法拉第保持架内。 注：这可以防止可能影响记录的机械振动和电气噪音。我们...

<ol>
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小龙虾由于能够在非自然条件下生存，已被证明是一个优秀的模型。易于进行体内和体外电生理分析。此外，甲壳类动物是比较时间生物学领域神经生物学研究的有利群体。
本文采用细胞内记录技术，对小龙虾光活化转导电流的脱敏和恢复进行了研究。然而，我们相信，只要有可能获得光受体细胞，同样的技术就可以适应其他无脊椎动物视觉?...

为了被视为视觉刺激，到达眼睛的光必须转换成电信号。因此，在所有视觉生物体中，光触发转导电电流，进而产生光受体细胞膜电位的变化，即所谓的受体电位。因此，眼睛的光灵敏度主要取决于光激活电导的状态，该电导可以激活或脱敏。
在小龙虾光感受器中，光触发一个缓慢的，瞬态的，离子电流1。照明后，转导电流在延迟或延迟后产生，然后达到最大值;之后，它衰减，因为转导通道进入脱敏状态，其中它们对进一步的光刺激2没有反应。也就是说，光除了激活对视觉负责的转导电流外，还会引起光受体细胞的灵敏度的瞬时递减。脱敏可能是防止过度暴露于适当刺激的一般保护机制。当转导电导从脱敏中恢复时，眼睛对光的敏感性会恢复。
细胞内记录是测量兴奋细胞3、4、5、6、7、8的电活性的有用技术。虽然随着贴片夹技术9的出现，细胞内记录已经变得不那么频繁，但当细胞难以分离，或者呈现几何形状，使得贴片夹紧的千兆密封的形成变得困难（即，贴片电极和抗电阻为109欧姆的膜之间的密封或紧密接触），它仍然是一种方便的方法。后者的例子是精子细胞10和本文研究的光受体细胞。根据我们的经验，Procambarus克拉克光感受器很难在初级文化中分离和保存;此外，它们是薄棒，使千兆海豹形成难以实现。在细胞内记录中，一个尖锐的电极被推进到细胞中，由周围的组织固定到位。电极被放大器的高速开关电路切碎，因此在电压脉冲之间采样电流。此模式称为不连续单电极电压夹（dSEVC 模式）11。电极的高电阻（小开口）阻碍细胞和移液器溶液之间的扩散交换，对细胞内环境产生最小的干扰。这种技术的一个潜在缺点是电极插入可能产生非选择性的泄漏电流;因此，必须小心避免从电池记录泄漏电流的大小可能会干扰预定的测量4，12。
在此，我们使用孤立的小龙虾眼眼，通过在电压夹条件下对光受体细胞进行细胞内电记录，评估光激活电导的脱敏和恢复。

<table border="0" cellpadding="0" cellspacing="0" style="width:644px;" width="644">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axoclamp2A&#160;</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td style="width:180px;">Amplifier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata 1200 Interface</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td>Digitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oscilloscope TDS430A</td>
			<td style="width:129px;">Tektronix</td>
			<td style="width:119px;"></td>
			<td>Analogic Oscilloscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Photostimulator PS33 Plus</td>
			<td style="width:129px;">Grass</td>
			<td style="width:119px;"></td>
			<td>Lamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puller PC-100</td>
			<td style="width:129px;">Narishige</td>
			<td style="width:119px;"></td>
			<td>Micropipette Puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puller P-97</td>
			<td style="width:129px;">Sutter Instruments</td>
			<td style="width:119px;"></td>
			<td>Micropipette Puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Capillary Tube Kimax-51</td>
			<td style="width:129px;">Kimble Products</td>
			<td style="width:119px;">34502</td>
			<td>0.8, 1.10, 100 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HS-2 Headstage</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td>Headstage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator MX-4</td>
			<td>Narishige</td>
			<td style="width:119px;"></td>
			<td>Mechanical Micromanipulator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereoscopic Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pClamp</td>
			<td>Axon Instruments Inc</td>
			<td></td>
			<td style="width:180px;">Data acquisition software for digidata 1200 interface</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Clampfit</td>
			<td>Axon Instruments Inc</td>
			<td></td>
			<td style="width:180px;">Analysis software linked to pClamp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Origin</td>
			<td>OriginLab Corp.</td>
			<td></td>
			<td style="width:180px;">Data analysis and graphing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">P-9333</td>
			<td>Minimum 99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Sulfate</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td>Minimum 99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">C5080</td>
			<td>Minimum 99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">H7523</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td>98.00%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hypochlorite solution</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">425044</td>
			<td style="width:180px;">Available chlorine, 10-15%&#160;</td>
		</tr>
	</tbody>
</table>

desensitization and recovery of crayfish photoreceptors upon delivery of a light stimulus

提出了一种研究小龙虾光感受器脱敏和回收的方法。我们使用不连续的单电极开关电压夹配置对隔离眼眼中的光受体细胞进行了细胞内电记录。首先，用剃刀刀片，我们在背角膜打开，以获得视网膜。之后，我们插入一个玻璃电极通过开口，并穿透一个细胞，如记录负电位报告。膜电位被夹在光受体的静息电位上，并应用光脉冲来激活电流。最后，采用两种光闪协议测量电流脱敏和恢复。第一个光闪光触发，在滞后期后，转导离子电流，达到峰值振幅后衰减为脱敏状态;以不同时间间隔应用的第二次闪光可评估光激活电导的状态。为了描述光激发电流的特征，测量了三个参数：1） 延迟（从光闪光传递到电流达到其最大值的 10% 之间的时间）;2） 峰值电流;3） 脱敏时间常数（电流衰减相的指数时间常数）。所有参数都受到第一个脉冲的影响。
为了量化脱敏回收，采用了 p2/p1 的比率与脉冲之间的时间。p1 是第一个光脉冲引起的峰值电流，p2 是第二个脉冲引起的峰值电流。这些数据被拟合到指数函数的总和上。最后，这些测量是作为昼夜时间的功能进行的。

首先，获得小龙虾光受体细胞的代表性受体电位（图4）。之后，应用了测试光闪光来触发光转导电流（图5）。阳离子转导电流1在滞后后激活，达到最大值，然后慢慢下降到吸收脱敏状态，从中缓慢恢复。
可以合理地假设，光激发电流的长延迟L（几十毫秒）取决?...

Watch this Scientific Journal Video about Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus at JoVE.com

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

提出了一种研究小龙虾光感受器作为昼夜时间函数的脱敏和灵敏度恢复的方案。

光刺激后小龙虾光受体的脱敏和回收

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

该程序的总体目标是研究小龙虾光感受器的脱敏和灵敏度恢复作为昼夜的一个函数，利用隔离眼库中光感受细胞的细胞间电记录，采用不连续的单电极开关电压夹紧配置。这种方法可以帮助回答神经生物学领域的关键问题，如光受体在灭活和恢复中许多电生理特性的昼夜时间依赖性。该技术的主要优点是，它允许测量光感受器的电活动。尽管难以分离细胞，不干扰细胞内环境。证明这个程序将是医生卡罗来纳·巴里加-蒙托亚和阿拉塞利德拉奥马里特内斯，我们实验室的同事。要准备细胞内电极，请用微管拉拔器拉取玻璃毛细管，以获得带小开口的薄尖端。接下来，用毛细管填充拉毛细管玻璃，用2.7摩尔氯化钾溶液填充，然后用细注射针填充一半的移液器。如有必要，点按电极移液器以消除气泡。之后，将电极放在支架上。使用放大器将支架连接到放大器头级。之后，使用稳定的 3-D 微操纵器定位电极支架头级。降低电极，直到沐浴溶液盖住其尖端。选择放大器的桥模并测量电极电阻。确保电阻约为 50 兆欧姆。使用放大器部分电压夹中的保持位置按钮和放大器微电位 1 部分中的电容中和按钮，将偏移电流静音并补偿电容瞬变。要构建超级熔合系统，请将浴液倒入合适的容器中，并连接到灌溉管组，从而连接记录室。使用重力驱动的超级输液系统。将流速调节到 0.5 毫升/秒。将腔室连接到吸气装置。使用真空泵调节溶液吸附系统，使记录室的总量不改变。要分离成年小龙虾的眼存量，请用一把细剪刀将其从底座上分离。用剃刀刀片在后角膜中打开一平方毫米，以进入视网膜。接下来，将眼部放在录音室的中心，视网膜朝上。在黑暗中保持眼睛存量20分钟。将微电杆平行于眼部垂直轴，使微电杆居中以进入视网膜。使用立体显微镜将设备置于正确的配置中。接下来，通过选择放大器的桥接模式，监控参考电极和记录电极之间的电压差。将电极放入浴缸中，并放在视网膜上。取下显微镜，将光刺激灯与眼部垂直轴平行放置。然后，慢慢降低微电机，直到检测到突然的电压下降。随后，提供测试光闪光，记录光感受器的受体电位。为了获得光引出电流的适当记录，对健康的光感受细胞进行良好的损伤至关重要。在此过程中，通过在数据采集软件中选择保持振幅，将电池的静膜电位固定电压。然后，选择放大器的 DSEVC 模式。在放大器的模式部分，选择 SEVC 按钮，然后将操纵杆切换到 Discont。SEVC 位置。将开关速率设置为 500 至 1，000 赫兹，由电极速度决定。之后，传递一个光闪光并观察唤起的离子通量。然后，传递一对光脉冲，并在所需的时间间隔后应用第二个闪光。使用数据采集软件以 10 千赫采样率对电流进行数字化，并保存数据以供离线分析。此处显示的是双脉冲协议。光刺激在零毫秒和700毫秒时应用。此图显示了从脱敏状态的恢复。这里是延迟恢复，这里是峰值电流恢复，这里是脱敏时间恒定tau恢复。实验在昼夜零时进行。此图显示了从脱敏作为昼夜时间函数的恢复。这是 iP 恢复，这是 L 恢复，这是 tau 恢复，此图显示加权时间常量。双相进程用箭头标记。一旦掌握，这项技术可以在两个小时内完成，如果它做正确。在尝试此过程时，重要的是要记住实现低噪声和低泄漏电流、光感受器损伤。遵循这个程序，其他方法，如药理学分析，可以做，以回答其他问题，如参与特定的通道，受体，或信号通路。该技术开发后，为神经生物学领域的研究人员探索光受体细胞电生理特性的昼夜时间依赖性铺平了道路。看完这段视频后，您应该对如何使用细胞内记录技术研究光激活转导电流的脱敏和恢复有了很好的了解。

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SCIENTISTS IN ACTION

Recording Behavioral Responses to Reflection in Crayfish

小龙虾的反思记录行为反应

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

科研

神经科学

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

小龙虾腿伸肌高和低输出NMJs生理录音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

小龙虾腹部肌肉受体机关：在本体的学生实验室练习

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

生理实验与小龙虾肠：学生实验室的工作

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

使用传统的空调和心率的方法刺激听觉声学和电刺激对歧视行为的测定

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

灭绝培训在彭兰霞窗口无法还原的恐惧

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

小龙虾的游泳足系统：实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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, ...