Name: levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions
Uploaded: 2018-05-05T14:00:00.000+00:00
Duration: 10 min 45 s
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><li>使用电动剃须刀去除头部、颈部和背部背面的大部分毛发.....。另外, 去掉头发周围和左耳朵。 注意: 在剃须耳朵周围的皮肤时要小心, 皮肤会被剃须刀的刀片夹住, 可能会损伤底层的肌肉组织。</li>
	<li>用棉签将脱毛膏涂抹在剃须部位, 除去剩下的毛发。等待约1分钟, 并用水洗瓶冲洗头发去除膏, 然后用棉签刷掉任何剩余的奶油或头发, 并轻拍皮肤干燥。</li>
</ol>3. 去除皮肤暴露提耳肌
<ol><li>在立体解剖显微镜下, 在肩胛的水平上做一个小切口 (只是深到足以穿透皮肤)。按照图 1A-b中显示的路径, 使用微解剖剪刀切割皮肤。</li>
	<li>使用精钳 (如5号), 拉起皮肤沿切口附近的肩胛。使用弹簧剪刀, 切断结缔组织, 使皮肤与底层肌肉组织在接近耳朵时分开。重要的是, 切断结缔组织与刀片指向皮肤, 以避免意外切割裸露的肌肉组织。</li>
	<li>定期灌注暴露的肌肉与生理盐水的解决方案, 如下面的 Na+外部缓冲区, 包括 (毫米): 144 氯化钠, 4 氯化钾, 1.2 CaCl2, 0.6 氯化镁2, 5 葡萄糖, 1 NaH2PO4, pH 7。4与氢氧化钠, 并有300的渗透压5毫摩尔/千克。一旦结缔组织被切断到左耳朵, 切断皮肤周围的耳朵, 以消除皮肤完全从鼠标和丢弃它。 注: 拉尔附着在耳朵的底座上, 在去除皮肤时容易受损。在耳底周围留下1毫米的皮肤是很好的, 以避免切割拉尔。</li>
</ol>4. 切除提耳肌和周围组织
<ol><li>使用弹簧剪刀, 首先切下的肌肉是低于拉尔, 它连接到脊柱之间的肩胛。从右肩胛骨开始, 就在中线的右侧, 并切向延髓端的鼠标, 同时保持在中线的右侧。继续切割所有的方式到年底的肌肉组织在头骨上。 注: 拉尔是连接到耳朵内侧和中线的最浅肌, 如图 2所示。对于其他图像, 由乳油格林17提供的一本书显示了拉尔的优秀手绘图像。</li>
	<li>使用镊子立即抓住切割组织在中线右侧, 然后轻轻地抬起, 并开始切割的组织向左耳朵与刀片的剪刀压在头骨上, 以消除几层肌肉, 低于左拉尔。暂时保留所附的所有层, 以避免在拆卸过程中意外地切割拉尔。</li>
	<li>切割与刀片平行, 并按对头骨, 以避免削减的神经, 支配的拉尔, 环绕耳道, 并进入肌肉的内侧一侧的耳朵。继续切割耳道, 保持尽可能多的神经连接。 注: 从脸部神经提供的拉尔分支的神经, 应保留, 因为它将在稍后的过程中使用。</li>
	<li>在耳朵的延髓部分的图 1B中显示的同一路径上, 切开刚刚穿过耳道的脂肪组织。另外, 沿着左肩胛骨切开, 就像在右侧一样, 完全从老鼠身上取出肌肉。</li>
</ol>5. 提肛耳肌分离
<ol><li>把拉尔和周围的组织放进一个容器里。使用可将解剖组织固定在底部的容器, 如带有硅胶弹性体底部的培养皿。沐浴并经常在生理盐水溶液中清洗组织。</li>
	<li>将耳朵的耳廓沿耳的底部切断, 使耳软骨部分附着在拉尔上。翻转肌肉, 使下等面朝上 (在盘子底部的拉尔)。</li>
	<li>放置一个针, 如昆虫别针, 通过耳道, 以保持准备到位。然后使用较小的针脚, 将其余的组织固定在拉尔中线的另一侧。使用镊子, 轻轻拉在耳朵的侧面部分的皮肤伸展肌肉, 并放置一个小针通过皮肤。重复此步骤, 直到将组织安全地放到菜上, 如图 3所示。 注意: 小解剖针可以通过切割针刺针的提示, 以达到预期的长度。这些小别针减少对组织的损伤, 并且可以使用与水浸泡显微镜目标以长的工作距离。</li>
	<li>开始移除肌肉 (如图 4所示), 它覆盖着拉尔 (耳针上级、外展人耳、和interscutularis), 以及那些通过结缔组织连接到拉尔, 在靠近中线, 使用5号钳和弹簧剪刀。</li>
	<li>使用镊子拉上上覆的肌肉层, 切断结缔组织与叶片定向到肌肉层被拉, 并注意避免切割或偷的拉尔。切向中线, 停止切割约三的道路中线。然后, 切割平行的中线, 以消除每个肌肉层。继续去除肌肉层直到只有拉尔留下。</li>
	<li>移除那些覆盖着拉尔的部分剩余的结缔组织, 这有助于电极的掷镖师掷。轻轻地使用5号钳把结缔组织从拉尔拉出来, 用弹簧剪刀把它剪掉。只有删除的组织, 可以这么容易地做到不损害的过程中的拉尔的危险。去除任何大的神经, 支配的下层肌肉层留在拉拉的下表面, 可能会阻碍神经肌肉结的可视化。</li>
</ol>6. 神经的分离
<ol><li>用神经刺激器识别支配的神经 (例如, 两条连接到脉冲发生器的白金导线);从耳道到肌肉会有好几个神经。用神经刺激器来接触神经, 提供一些电流 (电流将变化大约 5 V)。当肌肉收缩时, 正确的神经被识别出来。</li>
	<li>小心地抓住神经附近的组织, 用弹簧剪刀把神经从耳朵周围的组织中分离出来。为了最大限度地减少损伤, 把大部分的神经埋在一些周围的组织中, 这将被用来确保神经的记录碟。 注: 支配的运动神经位于耳道开口的内侧。</li>
	<li>如果使用双极刺激电极, 只需将周围的组织移除神经区域, 远离肌肉。如果使用吸入电极, 去除周围组织在神经的切口末端。 注意: 这是一个很好的停止点。这是一个很好的停车点。如果需要休息, 解剖的拉尔准备可以保持在一个生理的解决方案或持续灌注几个小时, 以确保有害的代谢物不积累。使用大量的溶液或恒定的灌注, 以确保有害的代谢物不会积聚。</li>
	<li>接下来, 脱离和转移肌肉到一个灌注室的电生理学实验下直立复合显微镜。 注: 使用可安全固定组织的软底灌注室 (图 5A)。</li>
	<li>将肌肉固定在末端 (耳朵和中线) 和沿肌肉的边缘。将神经垂直于肌肉纤维, 并将其钉在盘子的底部, 通过一些多余的组织在神经末端保持完好。在任何时候保持组织沐浴在一个生理生理盐水溶液中。 注: 直接在拉拉肌纤维下有圆形、高架材料也有帮助。这种额外的支持帮助在电极掷镖师掷, 可以由一个小段的硅胶弹性体铸造在50毫升圆锥管躺在它的一侧。弹性体的圆形截面可以用少量的新鲜硅胶固定在灌注室的底部。在图 5B-c中显示了灌注腔的示意图。肌肉纤维可以紧紧地粘在新铸造的硅橡胶 (这是非常疏水性) 和可能会损坏。这是有益的涂层或阻止新的铸造硅胶与蛋白质, 类似于阻止步骤在应用免疫印迹。为了阻止这种情况, 我们在一夜之间在牛血清白蛋白的浓缩溶液中孵化出新的硅胶。</li>
</ol>7. 电生理设备设置
<ol><li>准备或解冻整除数的染料, 以帮助可视化神经肌肉连接, 如线粒体染料 4-(4-diethylaminostyryl)-1-吡啶 18, 这是轻敏感, 应储存远离光.同时, 解冻电极溶液。涡旋所有整除数, 以确保所有溶质都在溶液中。准备一个含有1µM µ-芋螺毒素 GIIIB (µ CTX) 和80µM 基 (可选) 的生理盐水溶液。</li>
	<li>将带拉尔的灌注室固定在显微镜阶段。将参考电极放入装满3米氯化钾的杯中, 通过琼脂桥连接到记录室。根据制造商的指示将参考电极连接到放大器。</li>
	<li>此时, 将神经刺激电极放置在神经上。使用吸入电极或小两极电极, 因为它们能很好地用于神经刺激。<ol>
<li>将刺激电极放在远离肌肉的位置, 尽量减少录音中刺激工件的数量。 注: 刺激电极应控制与刺激隔离单元, 可以控制电压振幅必要的。</li>
	</ol>
</li>
	<li>通过打开刺激隔离装置上的电压旋钮, 慢慢提高电压, 直到观察到收缩。首先观察到收缩的点是阈值。确定阈值后, 将刺激隔离单元的电压设置为 1.5 * 阈值 (或由实验定义)。 注意:图 6显示了在直立显微镜下设置的肌肉准备, 以及使用球关节机械手在神经上定位的小两极电极。它是有利的为刺激有可能的最低的电压, 当仍然是充足地在门限之上。较低的刺激电压减少了录音过程中刺激工件的数量。另外, 将刺激电极放在远离肌肉的情况下, 会减少刺激工件的大小。有时神经的末端会从解剖中受损, 因此有必要在神经刺激器放置的地方进行实验。</li>
	<li>去除沐浴盐水溶液, 用5µM 4-二 2-Asp 替换。将拉尔准备的 4-2-Asp 为10分钟, 以获得足够的荧光神经肌肉连接可视化 (图 7)。</li>
	<li>准备两个电极溶液, 3 米氯化钾和 K+内部解决方案, 包括 (毫米) 75 天冬氨酸, 5 氯化镁2, 15 Ca (OH)2, 5 ATP 钠, 5 磷酸肌酸二钠, 5 谷胱甘肽, 20 拖把, 30 EGTA, pH 7.2 与 KOH。 注: 解决方案应事先准备好;K+内部解决方案可以存储在整除数-20 摄氏度。</li>
	<li>用3米氯化钾填充压敏电极的拉玻毛细管. 使用 K+内部解决方案 (在步骤7.6 中为当前通过的电极编写);使用一个狭窄的非金属针 (34 口径) 连接到1毫升注射器, 以方便地填补玻璃毛细血管。轻轻敲击毛细血管以除去气泡;确保每个填充电极的电阻为 10-15 MΩ。 注: 使用数据采集软件检查并注意两个电极的电阻。</li>
	<li>10分钟后, 使用常规 Ca2 +解决方案交换 4 Di Asp 解决方案。</li>
</ol>8. 用荧光识别神经肌肉结点
<ol><li>使用标准亮场和荧光照明的直立显微镜, 寻找沿准备方向垂直于肌肉纤维的荧光绿色神经肌肉连接的明亮带, 如图 7A所示。使用低放大水浸泡目标 (~ 10 x) 识别神经肌肉连接 注: 显微镜应配备 FITC 立方体 (前: 480/40, 分色: 505LP, Em: 535/50), 和 LED, 激光, 卤素灯, 或汞灯荧光。为了尽量减少照片漂白, 在明亮场和荧光照明之间交替。 注意: 这个波段通常是在耳朵的底部比中线更近。该波段是神经肌肉结最集中的区域。</li>
	<li>切换到更高的放大水浸泡目标 (~ 40 x), 并确定一个神经肌肉连接在顶层的肌肉检查与电生理学 (图 7B)。</li>
	<li>根据制造商的指示, 在适当的 headstages 上确保填充的吸管进入吸管持有者。使用并联在确定的神经肌肉连接的100µm 内将电极放置在肌肉膜上方 (图 7C)。使用主要的光明场显微镜定位电极;使用荧光来确认电极相对于神经肌肉连接的位置。</li>
	<li>首先, 使用低放大倍数 (~ 10 x) 目标定位电极, 然后切换到更高的放大倍数 (~ 40 x) 目标, 最终定位电极。此时不要将电极刺穿到肌肉中, 首先调整电极。</li>
</ol>9. 调谐和刺穿电极
<ol><li>一旦电极定位在所需光纤之上, 零点, 并通过桥平衡 (或类似的方法) 调整电压传感电极, 并将电极电容按制造商的说明中和。此外, 零电流通过电极。</li>
	<li>把两个电极都放到纤维表面。将电极的位置慢慢地调整到纤维上, 使电极保持与神经肌肉连接的方向, 如步骤8.3 和8.4 所述。</li>
	<li>两个电极接触到肌纤维的表面后, 先刺穿钝电流通过电极。使用诸如嗡嗡声 (超电容补偿的短暂脉冲)、短暂的电流脉冲或轻轻敲击桌子以方便电极掷镖师掷的技术。<ol>
<li>在数据采集软件中使用示波器或示波器协议监视信号, 直到识别出负膜电位, 这表明电极已被刺穿。</li>
	</ol>
</li>
	<li>将更锋利的电压感应电极压低到肌肉上, 直至刺穿电流通过电极的水平。一旦两个电极彼此一致, 刺穿的电压感应电极使用相同的技术应用与电流通过电极。</li>
	<li>在成功的掷镖师掷, 使用控制器为放大器, 应用一个恒定的负持有电流与电流通过电极, 以弥补任何膜损害, 由于电极掷镖师掷。当细胞开始慢慢向-80 mV 充电时, 根据需要增加保持电流, 使细胞达到预期的静止电位 (即,-80 到-85 mV)。 注意: 避免从纤维中记录在野生类型肌肉中需要比-25 nA 更大的保持电流, 以减少从不健康的纤维中记录的几率。</li>
	<li>一旦纤维稳定了几分钟的预期休息电位, 继续记录肌肉膜的性质或神经肌肉传播。</li>
</ol>10. 记录突触后膜特性和突触传输
<ol><li>从电流钳记录开始, 通过应用相等数量的正负电流步骤来测量相应的膜电压响应, 用于确定马达终板 R in 和τ m。要确保无源属性, 请使用达到稳定状态的小电压响应, 并具有线性电压电流关系16,19,20。避免从纤维上记录受损或不健康的神经, 这样的纤维有一个 mEPP 频率 > 3 赫兹。</li>
	<li>接下来, 根据制造商的说明输入双电极电压钳。压钳纤维在膜电位为-85 mV。电压钳设置应实现信噪比, 使检测 mEPCs。 注意: 保持适当的电压控制可能是一个问题。我们使用两种方法来最小化这些问题。首先, 纤维被刺穿在马达终板的100µm 之内如前面描述, 在这些纤维的长度常数之内21。其次, 我们使用放大器的 DC 恢复功能。此功能设置了非常高的电压钳增益。我们以前的工作详细描述了此方法, 并概述了一种评估电压箝位板的方法16。</li>
	<li>一旦在电压钳, 记录突触电流 (mEPCs 和 eEPCs) 使用各种神经刺激协议, 根据实验的要求。<ol>
<li>记录自发 mEPCs 和 EPCs 使用低频神经刺激协议 (≤0.5 Hz), 使一个记录 EPCs 没有突触调制 (促进或调制), 以评估量子内容。为了评估突触促进或抑郁症, 使用高频神经刺激协议 (10 脉冲在50赫兹)。</li>
		<li>通过一个刺激隔离单元刺激神经在期望的频率, 这可以被触发使用的脉冲发生器控制使用的数据采集软件。</li>
	</ol>
</li>
	<li>一旦电压钳录音完成, 返回放大器的电流钳设置, 关闭所有电流, 并从纤维中取出电极。检查在录音过程中电极没有在基线电压中堵塞或漂移。 注: 关于漂移, 电极电压应为0伏特的胞外溶液, 因为它们是在纤维掷镖师掷之前设置的。例如, 如果电压在实验过程中漂移, 则记录的电压是不确定的。</li>
</ol>

<ol>
	<li>Angaut-Petit, D., Molgo, J., Connold, A. 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作者没有什么可透露的。

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><tbody><tr><td>Olympus Compound Microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>10x Objective</td><td>Olympus</td><td>UMPLFLN10XW</td><td></td></tr><tr><td>40x Objective</td><td>Olympus</td><td>LUMPLFLN40XW</td><td></td></tr><tr><td>Borosilicate Glass</td><td>Sutter Instruments</td><td>BF150-86-7.5</td><td></td></tr><tr><td>CCD Camera</td><td>Santa Barbara Instruments Group</td><td>ST-7XMEI</td><td></td></tr><tr><td>Mater-9 Pulse Generator</td><td>AMPI</td><td></td><td></td></tr><tr><td>Iso-flex Stimulus Isolator</td><td>AMPI</td><td></td><td></td></tr><tr><td>pCLAMP 10 Data Acquisition and Analysis Software</td><td>Molecular Devices</td><td>1-2500-0180</td><td></td></tr><tr><td>Concentric Bipolar Electrode</td><td>FHC</td><td>CBDSH75</td><td></td></tr><tr><td>Ball-joint Manipulator</td><td>Narishige&amp;nbsp;</td><td></td><td></td></tr><tr><td>Non-metalic Syringes 34 Gauge</td><td>World Precision Instruments</td><td>MF34G-5</td><td></td></tr><tr><td>Nikon Stereomicroscope</td><td>Nikon</td><td>SMZ800N</td><td></td></tr><tr><td>No. 5 Forceps</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>No. 2 Forceps</td><td>Roboz</td><td>RS-5Q41</td><td></td></tr><tr><td>Microdissecting Scissors</td><td>Roboz</td><td>RS-5912SC</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>2404019862</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Grass SD9 Stimulator</td><td>Grass Medical</td><td></td><td></td></tr><tr><td>Model P-1000 Micropipette Puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>Axon Digidata 1550 Low-noise Data Acuisition System</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Low Pass Bessell Filter</td><td>Warner Instrument Corp.</td><td>LPF-8</td><td></td></tr><tr><td>Left-handed Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641/45DL</td><td></td></tr><tr><td>Right-handed Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641/45DR</td><td></td></tr><tr><td>Single Motion Controler</td><td>Siskiyou Corp.</td><td>MC100e</td><td></td></tr><tr><td>Crossed Roller Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641R</td><td>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><td>All chemicals were orded from Fisher except,</td><td></td><td></td><td></td></tr><tr><td>BTS</td><td>Toronto Research Chemicals</td><td>B315190</td><td></td></tr><tr><td>CTX</td><td>Alomone Labs</td><td>C-270</td><td></td></tr><tr><td>4-Di-2-Asp</td><td>Molecular Probes</td><td></td><td>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...

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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 ...

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

9.3K 次观看.  Wright State University. 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。

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

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

科研

生物学

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

神经科学

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

了解运动神经元对脊髓刺激的反应

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

作者聚焦:推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

摘要

探索更多视频

此视频中的章节

电生理方法记录从果蝇幼虫NMJ突触潜力

线虫神经肌肉交界处的解剖和录制

在电生理记录果蝇胚胎

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

一

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

摘要

探索更多视频

此视频中的章节

电生理方法记录从果蝇幼虫NMJ突触潜力

线虫神经肌肉交界处的解剖和录制

在电生理记录果蝇胚胎

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

一

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录