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。
<ol><li>
使500毫升的额外细胞溶液没有 ca2 + (ca2 +自由 EC): 150 毫米氯化钠, 5 毫米氯化钾, 1.3 毫米氯化镁2, 4 毫米葡萄糖, 10 毫米 Hepes。
	<ol>
<li>溶解 4.38 g 氯化钠, 186.37 毫克氯化钾, 61.89 毫克氯化镁2, 360.32 毫克葡萄糖和1.19 克 HEPES 在450毫升超纯 (0.055 美国/厘米在25°c, 电阻率18.2 毫欧) H2O 在玻璃烧杯使用磁力搅拌器。</li>
		<li>用氢氧化钠将 pH 值调整为7.75。</li>
		<li>将溶液转化为500毫升的容积烧瓶。用超纯 H2O 填充体积瓶, 直到500毫升。</li>
		<li>在测量渗透压之前, 先把溶液搅拌好。用甘露醇调节 290–300 mOsm。过滤器用水真空系统和0.2 µm 过滤器消毒溶液。</li>
	</ol>
</li>
	<li>
使500毫升的额外细胞溶液 (EC): 150 毫米氯化钠, 5 毫米氯化钾, 2 毫米 CaCl2, 1.3 毫米氯化镁2, 4 毫米葡萄糖, 10 毫米 Hepes。
	<ol>
<li>用磁力搅拌器在玻璃烧杯中溶解4.38 克氯化钠、186.37 毫克氯化钾、147.01 毫克 CaCl2、61.89 毫克氯化镁2、360.32 毫克葡萄糖和1.19 克 HEPES 在450毫升的超纯 H2O。</li>
		<li>用氢氧化钠将 pH 值调整为7.75。</li>
		<li>将溶液转化为500毫升的容积烧瓶。用超纯 H2O 填充体积瓶, 直到500毫升。</li>
		<li>在测量渗透压之前, 先把溶液搅拌好。用甘露醇调节 290–300 mOsm。过滤器用水真空系统和0.2 µm 过滤器消毒溶液。</li>
	</ol>
</li>
	<li>
用0.1% 牛血清白蛋白 (EC 与 BSA) 制作500毫升的超细胞溶液: 150 毫米氯化钠, 5 毫米氯化钾, 2 毫米 CaCl2, 1.3 毫米氯化镁2, 4 毫米葡萄糖, 10 毫米 Hepes。
	<ol>
<li>用磁力搅拌器在玻璃烧杯中溶解4.38 克氯化钠、186.37 毫克氯化钾、147.01 毫克 CaCl2、61.89 毫克氯化镁2、360.32 毫克葡萄糖和1.19 克 HEPES 在450毫升的超纯 H2O。</li>
		<li>用氢氧化钠将 pH 值调整为7.75。</li>
		<li>将溶液转化为500毫升的容积烧瓶。用超纯 H2O 填充体积瓶, 直到500毫升。</li>
		<li>在测量渗透压之前, 先把溶液搅拌好。用甘露醇调节 290–300 mOsm。过滤器用水真空系统和0.2 µm 过滤器消毒溶液。添加50毫克 BSA。</li>
	</ol>
</li>
	<li>
使250毫升胞内溶液 (IC): 110 毫米 KOH, 20 毫米氯化钾, 10 毫米 Hepes, 20 毫米蔗糖。
	<ol>
<li>用磁力搅拌器在玻璃烧杯中溶解1.54 克 KOH, 327.75 毫克氯化钾, 595.75 毫克 HEPES 和1.712 克蔗糖, 在450毫升的超纯 H2O 中。</li>
		<li>用 (n-吗啉代) 乙烷磺酸 (MES) 将 pH 值调整为7.2。</li>
		<li>将溶液转化为250毫升的容积烧瓶。用超纯 H2O 填充体积瓶, 直到250毫升。</li>
		<li>在测量渗透压之前, 先把溶液搅拌好。调整到 280–290 mOsm (10 mOsm 低于 EC) 与蔗糖。过滤器用水真空系统和0.2 µm 过滤器消毒溶液。</li>
	</ol>
</li>
	<li>在 ca2 +和 BSA 自由 ec (2 克低熔融琼脂糖在100毫升的钙2 +自由 ec) 制造100毫升2% 低熔融琼脂糖溶液。用微波炉溶解, 将融化的琼脂糖放在40摄氏度的水浴中。让它冷却下来, 直到它达到40°c 之前使用的组织。 注: ec, Ca2 +免费 EC 可以储存在4摄氏度, 如果无菌和 IC 解决方案在-20 摄氏度, 几个月。琼脂糖可以储存一个星期在4°c, 可以重复使用3–5时间简要微波。过量再利用会导致琼脂糖的蒸发和改变, 盐的浓度和质量。</li>
	<li>
制作琼脂桥:
	<ol>
<li>热7.5 毫米长的硼硅酸盐玻璃毛细血管与外径 (外径) 2 毫米和内径 (id) 1.16 毫米使用本生燃烧器在一个焦点约1/3 从一端直到玻璃开始弯曲。从火焰中取出玻璃, 并允许玻璃以大约120度的角度弯曲 (图 2A)。</li>
		<li>在正常 EC 中, 用微波炉融化2% 琼脂, 而琼脂糖则是液体, 用毛细现象力将玻璃的一端放进液体中, 等待几分钟。商店桥梁, 直到使用4°c 在 EC 没有葡萄糖。</li>
	</ol>
</li>
	<li>用合适的吸管拉吸管用灯丝贴片制备硼硅酸盐玻璃。请参阅吸管牵引器手册, 以获得所需的电极电阻。 注: 吸管的锥度应尽可能短。采用第4-6 拉步法实现了短吸管锥度。电极电阻应介于2和 6 MΩ之间, 具体取决于细胞大小。</li>
	<li>
为了避免组织在膜片钳实验中的运动, 在硼酸盐缓冲器中涂上0.1% 聚乙烯亚胺 (裴) 的记录腔表面。
	<ol>
<li>准备500毫升硼酸盐缓冲器 (25 毫米):<ol>
<li>用磁力搅拌器在玻璃烧杯中溶解4.768 克 Na2B4o7/10H2o 的450毫升的超纯 H2o。</li>
			<li>用 HCl 将 pH 值调整为8.4。</li>
			<li>将溶液转化为500毫升的容积烧瓶。用超纯 H2O 填充体积瓶, 直到500毫升和0.2 µm 过滤器用水真空系统消毒溶液。</li>
		</ol>
</li>
		<li>在25µL 的硼酸盐缓冲器中, 通过稀释0.1% µL 10 个库存溶液, 将1% 的裴 (1 毫升50% 裴至49毫升1% 毫米硼酸盐缓冲器) 和100裴稀释剂准备好, 最后使用。</li>
		<li>在片架上的玻璃上添加2毫升0.1% 的裴, 让涂层孵化1分钟。然后用5毫升的超纯 H2O 简单地洗两次玻璃, 让空气干燥, 直到使用。</li>
	</ol>
</li>
	<li>
准备两性霉素 B 溶液。
	<ol>
<li>60毫克/毫升的股票溶液溶解3毫克的两性霉素 B 粉在50µL 二甲基亚砜 (亚砜), 在1毫升管保护免受光。漩涡在最大速度三十年代和油脂实验15分钟到融汇。储存整除数到3–5管在摄氏-20 摄氏度和使用一个整除每天。 注: 如果两性霉素 B 的股票溶液不清晰, 黄色, 但乳白色 (仍然黄色) 后 15–20 min 超声波做一个新的股票解决方案。</li>
		<li>吹打2毫升的 ic, 使 ic 溶液与两性霉素 B 结合成一个干净的玻璃烧杯, 容量超过10毫升。在吸管溶液中加入8µL 的两性霉素 B 溶液, 并用吹打混合。用铝箔包裹烧杯, 放在冰上, 每3小时更换一次。</li>
	</ol>
</li>
</ol>2. Vibratome 的解剖和切片
<ol><li>解剖前准备解剖工具, 包括一个锋利的和一个强钳与剪刀。用乙醇清洁工具 (纸巾夹、刷子、镊子), 并确保它们仅用于解剖目的, 而不与固定组织接触。把它们放在一个单独的盒子里以避免污染。</li>
	<li>弄死冰水中的鱼1分钟。</li>
	<li>
按照随后的步骤, 快速解剖鳉的大脑和垂体与锋利的钳 (使用不超过3分钟)。
	<ol>
<li>用强钳抓鱼时, 用剪刀或尖钳把眼睛后面的两个视神经都严重。</li>
		<li>将颅骨的背部从后向前侧打开, 用强钳一步步折断颅骨。</li>
		<li>用坚固的镊子从一侧剥离头骨。</li>
		<li>用剪刀或锋利的镊子把脊髓严重的。</li>
		<li>轻轻地, 抓住脊髓与镊子, 翻转大脑从后到前, 并把它放入一个菜与冰冷 Ca2 +免费 EC。</li>
	</ol>
</li>
	<li>用液体琼脂糖填充一个 1.5–2 cm3金属模子并且放置它在冰上。</li>
	<li>就在琼脂糖凝固之前 (在25–30°c 附近), 用镊子轻轻地抓住大脑, 用一张细纸擦干镊子, 把纸巾放进琼脂糖里。快速混合琼脂糖, 稀释在组织周围留下的欧共体的痕迹, 并定位组织, 让模具在冰上几秒钟, 直到琼脂糖硬化。 注意: 这一步必须快速。当金属模被冰冷却后, 琼脂糖很快就凝固了。</li>
	<li>用手术刀刀片修剪含有组织的一方形琼脂糖, 并用手术 (无毒) 胶水将其粘在 vibratome 标本持有者身上。</li>
	<li>填充试管的 vibratome 接受标本 (脑垂体) 持有人与冰冷的 Ca2 +免费 EC。</li>
	<li>将标本持有者放入 vibratome 中, 并切开琼脂糖块, 使旁部分150µm, 使用高频和低速切片。收集选定的部分, 并将其放入膜片钳记录室与3毫升冰冷 Ca2 +免费 EC。</li>
	<li>将网格竖琴 (图 2B) 放在组织部分。 注: 竖琴将与涂层稳定的组织和防止它移动在补丁钳录音。</li>
	<li>在竖琴到位后, 将 ca2 +免费 ec 培养基改为正常 ec 与 ca2 +和 BSA。让纸巾休息10分钟。</li>
</ol>3. 穿孔膜片钳和电生理记录
<ol><li>在开始实验之前, 氯化银线电极由以下步骤: 首先, 使用细砂纸 (p180) 清洁银线电极。第二, 用2毫升, 70% 乙醇冲洗。第三, 将导线浸在2毫升, 2–5% 氯为10分钟。最后, 用2毫升, 超纯 H2O 冲洗。</li>
	<li>将记录室与脑垂体切片放置在显微镜阶段, 用10X 目标定位目标区域 (垂体), 并使用40X 目标对细胞进行检查。 注意: 如果准备工作成功, 则很少有圆形细胞可见。这些圆形细胞通常被损坏的细胞从组织中分离出来。</li>
	<li>将接地电极通过2% 琼脂桥进入欧共体浴。</li>
	<li>使用40X 目标定位健康细胞。 注意: 一个健康的细胞应该牢固地附着在组织切片上, 而不是从切片中向上和分离。</li>
	<li>在电压钳中设置放大器 (VC;图 4A点 1) 模式。打开密封测试窗口并按下浴缸配置 (图 4B点1和2。使用 5 mV 脉冲监测吸管阻力 (图 4B点 3)。</li>
	<li>用微填料注射器在用两性霉素 B 添加 ic 溶液之前, 用无真菌的 ic 溶液将贴片吸管的尖端回填 (见图 3)。</li>
	<li>使用1毫升注射器在贴片吸管中添加轻微的正气压。 注意: 这使得一个小的液体流动的补丁吸管, 避免污染的尖端。</li>
	<li>一旦在浴缸中, 评估贴片阻力, 并确保没有颗粒附着在吸管的尖端 (图 4C)。 注意: 将一小块磁带连接到显示器上, 显示视频录制形式的视野。这使得当目标不专注于细胞时, 贴片吸管相对于细胞更容易定位。</li>
	<li>使用并联将贴片吸管向下引导至细胞。 注意: 通过寻找可能附着在吸管尖端的小颗粒来确保吸管是干净的。阻力的突然变化也可能是在吸管尖端的粒子卡住的结果。</li>
	<li>调整吸管, 当几个微米以上的细胞, 使吸管的尖端将接触细胞1/3 从细胞中间, 如图 5所示。当触摸细胞, 释放压力, 并应用温和的吸力, 使印章。 注: 从贴片吸管进入浴缸的时间应尽可能保持短。不超过1–1.5 分钟, 以避免两性霉素 B 到达吸管的尖端和泄漏到浴缸。</li>
	<li>立即切换到修补程序窗口中的补丁钳软件 (图 4D点 1), 并有一个持有的潜力在-50 和-60 mV (图 4D点 2)。零出的快速电容由玻璃吸管 (图 4D点 3)。</li>
	<li>切换到补丁钳软件中的单元格窗口 (图 4E点1和 2), 并开始监视访问电阻, Ra, 显示在窗口上。将放大器软件中的膜电容降至零 (图 4E点 3) 经过足够的访问。 注意: 足够的访问可能需要10到30分钟 (图 4E点 2)。对当前钳位记录的良好访问应低于 20 m。</li>
	<li>切换到电流钳, IC, 放大器软件窗口 (图 4F点 1)。调整放大器软件的I 钳窗口中的快速电容电流 (图 4F点2和 3), 与3.12 中的值相同。检查膜电位 (图 4F点 1)。 注意: 重要的是, 如果膜电位很浅 (典型的在-35 mV 以上), 细胞可能会受损。取消录制并查找新单元格。</li>
	<li>在找到一个健康的细胞 (牢固地附着在膜电位低于-40 mV 的组织), 开始实验记录, 详细的制造商的协议33,34。</li>
</ol>

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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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			<td style="width:157px;">BF200-116-15</td>
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			<td height="21" style="height:21px;width:309px;">BSA</td>
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			<td style="width:157px;">A2153</td>
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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 ...

healthy-brain-pituitary-slices-for-electrophysiological

Research

JoVE Journal

Neuroscience

7.7K 次观看.  Norwegian University of Life Sciences. 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。

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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

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" ...

科研

神经科学

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

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

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

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

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 ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

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 ...

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

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

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

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

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

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

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 ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

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

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

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 ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由游泳鱼中可移动的特龙对神经元的无线电生理记录

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...