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。

<table><tbody><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>Chirurgical glue</td><td>WPI</td><td>VETBOND</td><td>3M Vetbond Tissue Adhesive</td></tr><tr><td>Stainless steel blades</td><td>Campden Instruments</td><td>752-1-SS</td><td></td></tr><tr><td>metal molds</td><td>SAKURA</td><td>4122</td><td></td></tr><tr><td>steel harp</td><td>Warner instruments</td><td>64-1417</td><td></td></tr><tr><td>PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Ultrapure LMP agarose</td><td>invitrogen</td><td>166520-100</td><td></td></tr><tr><td>patch pipettes</td><td>Sutter Instrument</td><td>BF150-110-10HP</td><td>Borosilicate with filament O.D.:1.5 mm, I.D.:1.10 mm</td></tr><tr><td>Microscope Slicescope</td><td>Scientifica</td><td>pro6000</td><td></td></tr><tr><td>P-Clamp10</td><td>Molecular Devices</td><td>#1-2500-0180</td><td>sofware</td></tr><tr><td>Digitizer Digidata 1550A1</td><td>Molecular Devices</td><td>DD1550</td><td></td></tr><tr><td>Amplifier Multiclap 700B Headstage CV-7B</td><td>Molecular Devices</td><td>1-CV-7B</td><td></td></tr><tr><td>GnRH</td><td>Bachem</td><td>4108604</td><td>H-Glu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-OH trifluoroacetate salt&amp;nbsp;</td></tr><tr><td>pipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td></td></tr><tr><td>amphotericin B</td><td>SIGMA</td><td>A9528</td><td>pore-forming antibiotic</td></tr><tr><td>polyethylenimine&amp;nbsp;</td><td>SIGMA</td><td>P3143</td><td>50% PEI solution</td></tr><tr><td>microfiler syringe</td><td>WPI</td><td>MF28/g67-5</td><td></td></tr><tr><td>glass for the agar bridge</td><td>Sutter Instrument</td><td>BF200-116-15</td><td>Borosilicate with filament O.D.:2.0 mm, I.D.:1.16 mm Fire polished</td></tr><tr><td>Micro-Manager software</td><td></td><td></td><td>Open Source Microscopy Software</td></tr><tr><td>optiMOS sCMOS camera</td><td>Qimaging</td><td>&amp;nbsp;01-OPTIMOS-R-M-16-C</td><td></td></tr><tr><td>sonicator</td><td>Elma</td><td>D-7700 singen</td><td></td></tr><tr><td>NaCl</td><td>SiGMA</td><td>S3014</td><td></td></tr><tr><td>KCl</td><td>SiGMA</td><td>P9541</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SiGMA</td><td>M8266</td><td></td></tr><tr><td>D-Glucose</td><td>SiGMA</td><td>G5400</td><td></td></tr><tr><td>Hepes</td><td>SiGMA</td><td>H4034</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SiGMA</td><td>C8106</td><td></td></tr><tr><td>Sucrose</td><td>SiGMA</td><td>84097</td><td></td></tr><tr><td>D-mannitol</td><td>SiGMA</td><td>63565</td><td></td></tr><tr><td>MES-acid</td><td>SIGMA</td><td>M0895</td><td></td></tr><tr><td>BSA</td><td>SIGMA</td><td>A2153</td><td></td></tr></tbody></table>

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

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

科研

神经科学

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

用心脏灌注和二固定仪对脑和垂体血管进行标记

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...

生物工程

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

从鱼类垂体制备优质原代细胞培养

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

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 ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

<meta charset="utf8"></head><body>从成年啮齿动物的海马背极制备急性脑切片

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

从成年啮齿动物的海马背极制备急性脑切片

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

健康脑-垂体切片对硬骨鱼类鱼垂体细胞电生理的研究

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此视频中的章节

膜片钳电容测量和Ca^{2 +}单神经末梢成像在视网膜切片

斑马鱼幼虫的前脑的电生理记录

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

在完整的成年斑马鱼大脑电生理记录

在脑片全细胞膜片钳记录

用心脏灌注和二固定仪对脑和垂体血管进行标记

从鱼类垂体制备优质原代细胞培养

视网膜原酸和皮质化突触功能的电生理学研究

从成年啮齿动物的海马背极制备急性脑切片

从成年啮齿动物的海马背极制备急性脑切片