罗纳德L. Calabrese，迪特尔积（埃默里大学）和沃德Yuhas（Axon仪器）有用的技术讨论，我很感谢。

<ol><li>下丘脑脑片准备，培养和转移以前详细的录音室（9）在动物的GnRH神经元表达GnRH的肽子（10）控制下的绿色荧光蛋白（GFP）。从动物的大脑取出后，大脑是用刀片，以消除不感兴趣的地区封锁。为了确定删除的地区，大脑是放置在其背水面，使下丘脑可以可视化。对于这两种冠状​​面和矢状面片的方向，小脑被删除。冠状切片准备，皮层延髓部分将被删除。在这片取向，皮层外侧地区提供支持大脑切片和银线的位置，以确保在录音室的大脑，大脑提供足够的面积。矢状切片准备，大脑外侧地区和皮质延髓部分提供的脑切片和银线的位置，以确保在录音的大脑以及支持。整个阻断过程中，大脑是不断蘸冷人工脑脊液中使用的玻璃吸管（学联）。 </li><li>少量的强力胶放在切割平台上。大脑是用抹刀解除。超额学联带来一个Kimwipe锅铲表面和绘图过剩学联从大脑中删除。大脑是轻轻滑落到胶水。触摸大脑或推下来，以确保它是很重要的。大脑本身的重量通常是足够的，以确保它是安全的。 </li><li>大脑和切割平台上，然后固定在振动切片机和切割以及充满冷学联。寒冷的解决方案两家公司的大脑，并可能在切割过程中不受损坏保护。在寒冷的程度，优化质量片不同的大脑区域不同。编制下丘脑薄片学联温度一般在0 ° C </li><li>应缓慢切割片，每个切片从大脑，应该自由浮动。如果片开始卷曲在切割过程中，刀片正在推进的速度应放慢。我们可以使用一个将其展平片的小油漆刷，但这样做风险损害的切片，因此，应尽量避免。慢慢刀片的前进速度优于使用油漆刷。 </li><li>由于每个切片切断从大脑，它是从切割室撤回，并放置在一个温水浴（约32 ° C）的片孵化室。切片孵化器不断提供与使用的95％O 2和5％的CO 2的氧气的混合物。一般情况下，预录片潜伏期从30分钟到2小时不等。 </li><li>玻璃吸管（3-6MΩ）制造如前所述（9）使用垂直拉拔。拉动的持续时间和温度变化的基础上的吸管拉马使用，玻璃使用的加热丝的拉拔寿命。枪头的形状可以根据用户的喜好也各不相同。一个0.1毫米的开放统一的锥形尖，我们已经取得了良好的成功。在吸管的开口更大，往往会损害神经元，而较小的开口失败导致持续时间长的录音。 </li><li>涂与Sylgard移液器，以减少电容。拉枪头轻轻涂抹Sylgard 184使用第二个吸管。 Sylgarded提示，然后用热风枪加热固化。这些移液器充满浴灌流。为了增加可视化的枪头，加入少量的Alexa - 568液用于灌装前的移液器移液器解决方案的一部分。我们不重量的Alexa - 568而是使用足以让眼睛的解决方案，粉红。枪头，然后可以根据得克萨斯州红色滤光片显示。 </li><li>我们已经开发出一种方法来获得电流钳检测使用低阻力密封件（见讨论）的行动潜力。内源性细胞的静息电位没有适用于目前使用的Axon仪器的2B Axoclamp录音。但是，由于低电阻密封，不能只用2B放大器检测动作电位。为了提高信号，第二个放大器（AM系统3000）是用于与Axoclamp 2B系列。这种方法有松动的密封和长期的唱片实用方便。从技术上讲，可以直接测量动作电位，从而规避在电压钳记录模式与方法的问题（见讨论）。 </li><li>一个需要两个移液器以前选定的两个神经元的表面在同一时间，在类​​似的方式作为对全细胞记录配置的方法。走近两个NE同时urons已比较成功的，在获得独立不是试图对每一个神经元的双唱片。在这段时间内，注入电流脉冲通过从2B放大器在全细胞记录的方法进行。 </li><li>都移液器必须保持正压，以防止堵塞尖。最好使用悬空3毫升空注射器连接管件的长度吸管​​持有人产生正压双录音。使用注射器，允许上都移液器保持压力。正压应尽快移液器一直在录音室的液放在应用。一种方法移液器顺序选定的神经元。定位后最大的神经元表面的第一个吸管直接，保持积极的留在注射器和叶子在这个位置上的吸管。然后返回到录音室的顶部和降低位置的第二个记录吸管。选择神经元往往是在不同层次的切片。第一移液器应定位在神经细胞最深的是在切片和更肤浅的神经元位置的第二个吸管。这可以防止转向第二吸管定位片将在垂直平面内移动略有移液器进入片的第二个吸管定位。 </li><li>松散密封（18-30MΩ）将被使用。正压力将促使每个细胞的表面上的一个小酒窝。人们试图释放正压和应用非常轻微的负压密封的第一个神经元。正压释放取出注射器。经口吸气，然后将其应用于油管的自由端。一个健康的神经元的膜将迅速做出反应释放的正压和坚持移液器。轻微的吸力将形成一个密封在一个健康的神经元的适当性。 </li> <li>尝试当一个密封的神经元和失败，吸液管不能被重新使用，因为膜只密封非常干净的玻璃。相反，提高移液器（物镜）离片表面的灌注井的顶部，从浴缸中删除的吸管，干净的移液管的变化，然后返回到切片试图密封另一个单元格。基于这些原因，重要的是要选择多个录音潜在的神经元。对于片不少于4-6录音质量的神经细胞，从中选择，获得双录音的成功是有限的。 </li><li>建立一个松散的密封电阻后，从2B放大器的电流注入脉冲终止​​。 2B amplifer是摆在系列与3000系列amplifer。在此配置中，2B amplifer的输出作为输入为3000系列amplifer。后者amplifer提供数据采集的信号。 </li><li>然后重复密封（步骤4）amplifers的重新配置（步骤5）的第二个神经元。 </li></ol><img src="/files/ftp_upload/1678/1678fig1tmb.jpg" alt="图1" border="0" /> 图1。一个时期，增加射击两个GnRH神经元在矢状切片准备宽松的密封连接的记录配置。片是来自一个阉割的男性。每向上偏转表示动作电位。注意GnRH的神经元（顶部的痕迹）展品几乎连续发射，而第二GnRH的神经元具有间歇性的阵阵。这种模式从单一的GnRH神经元的活动期间（7）体内的激素分泌多单元录音中提取的单一单位。请<a href="https://www.jove.com/files/ftp_upload/1678/1678fig1.jpg">点击这里</a>看到图1的放大版本。

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活动发生在一些包括GnRH神经元的神经元（基础上激素分泌）的利息小时的时间尺度（5-7）。因此，整个单元的配置，是不是一些由于在细胞内的全细胞记录模式使者的透析的实验目标的最佳选择。其次，一般仅限于动物比大约120天的年龄的全细胞记录。随着年龄的增长，出现神经细胞膜变硬，使得难以实现的高抗密封。此外，如果获得了高抗封，破裂修补破坏密封，留下了一个洞，吸液管和膜之间。这将导致无法使用的录音和神经元离子不平衡，很快就会死于。正常卵巢周期，因此，GnRH脉冲发生器的稳定的活动不会发生，直到以后的生活中超越年龄（7-10个月的年龄在C57BL6女性; 11日，12），当一个人可以合理预期获得全细胞记录可靠。最后，全细胞记录销毁内部和外部的离子浓度的内源性的比率。全细胞记录，内部的任何离子浓度等于在吸液管溶液的离子浓度。这是因为液的量较大，迅速达到平衡，与/替换内源性细胞的体积相对较小。 松散的细胞连接的方法绕过全细胞记录的许多限制。首先，低阻力密封（15-30MΩ）都可以使用。这些都是比较容易形成，即使在老年动物的神经元。其次，不破裂的膜密封补丁。因此，松散的细胞附着录音技术上远远高于全细胞记录更容易。此外，由于细胞膜是完整的，细胞内的组成部分透析也不会发生内源性离子比率保留。不能用于研究突触电流松散的细胞连接的方法，但它是理想的神经元在一个相对非侵入性的方式，从长期的录音。细胞附着的录音也可以使用吸管在细胞内任何标准的解决方案。这提供了额外的好处膜修补破裂的长期录制完成时，与细胞内的标记和标签神经元。 松散的细胞连接的方法已被用于在电压钳记录模式。然而，电压钳记录在宽松的细胞连接的配置有几个方法论问题。首先，记录的信号是一个活动的间接测量。测量的信号（如所谓的动作电流） 的电容电流，收费膜（13） 。这是一个极其重要的方法论问题。电容和电阻的一个记录吸管可以过滤记录的信号。这是非常可能的，小的动作电流在充电，不能正常与大多数放大器，由于探头的高电阻补偿吸管的电容丢失。当这些信号不被发现的，明显的神经元放电模式并不能反映真实的射击模式。同样地，无偿吸管和密封电阻，在测量过程中的重大变化时，如动作电流表示（13）错误。有些放大器可提供电容和电阻“补偿”的吸管和密封，这限制了信号的损失，但大多数放大器的高阻抗头阶段阻碍了最佳补偿。其次，一个人造的情况是对细胞施加。在电压钳模式下，细胞膜周围地区举行一个定势，在这些研究中，0 mV时。这并不意味着有目前没有适用于细胞膜。在电压钳测得的信号实际上是适用于膜，以保持定势的电流量。因此，这种施加的电流可以改变细胞的活性。 GnRH的系统中的双唱片特别具有挑战性由于GnRH神经元和他们的弥漫性分布的数量有限。对于双录音是成功的，操纵者必须极其稳定。即使是轻微的电极运动，可以导致移液器滑落的神经元和停止录音。此外，移液器对细胞的运动（例如，重新定位，以弥补运动）可以改变射击模式。机械敏感的一些离子通道等N型钙通道是：拉伸膜原因重复性活动，在全细胞和细胞贴附记录配置（14）。最后，操纵系统必须能够极为细腻的动作。如上所述双录音，需要两个移液器以前选定的两个神经元的表面密封在同一时间，并尝试一个神经元。如果成功的话，那么人企图封第二个单元格。一般来说，人们不能指望密封，并与每一个尝试高品质录音。然而，这创建一个具有双重录制的特定问题。如果一个人成功的第一个神经元，但第二次失败，一个必须改变的吸管，并尝试不同的细胞。因此，人们必须能够移动而不破坏的成功密封的神经元都在显微镜的浸泡目标和吸液管灌注井的顶部（变化移液器）。 我们的发展和使用的松散的细胞贴附双录音的方法是研究GnRH神经元的一个重大的技术进步。这是可能产生有用的结果，这将有助于在什么样的机制协调活动背后的关键问题背景下，移动领域向前搏动性荷尔蒙分泌的结果。

dual somatic recordings from gonadotropin-releasing hormone (gnrh) neurons identified by green fluorescent protein (gfp) in hypothalamic slices

促性腺激素释放激素（GnRH）是一个小的神经肽的调节垂体释放促黄体激素（LH）和促卵泡激素（FSH）。这些促性腺激素的生殖功能的调节重要。弥漫的地方，他们从轴突末端释放到hypophysiotropic门户系统（1）促性腺激素释放激素GnRH的含神经元分布，整个下丘脑和项目正中隆起。在门户毛细血管，GnRH的垂体前叶促性腺激素释放进入全身循环，以刺激。 GnRH的释放是不连续的，而是在发生偶发脉冲。这是公认GnRH的释放的间歇性地再现（2，3）是必不可少的。 多个GnRH神经元可能基础GnRH的脉冲活动的协调。 GnRH神经元的总肽含量约1.0皮克/细胞（4），其中30％可能包括释放的池。 GnRH的脉冲期间（5，6）的水平，建议多个GnRH神经元可能参与神经分泌。同样，单一单位的活动中提取LH释放期间从下丘脑多单元录音显示多个神经元的活动（7）的变化。要么GnRH的somata或纤维（8）与LH脉冲活动期间记录的电极。因此，这项活动至少部分来自GnRH神经元。 下丘脑GnRH神经元同步发射的机制不明。阐明GnRH神经元发射的机制，协调是一个复杂的问题。首先，GnRH神经元的数量相对较少。在啮齿类动物中，有800-2500 GnRH神经元。目前尚不清楚，情节GnRH的释放GnRH神经元。此外，GnRH神经元弥漫性分布（1）。这有复杂的射击协调的认识，并取得了许多技术方法棘手。我们已经优化了在电流钳模式动作电位的直接检测细胞连接松散的录音，并开发出了录音的方式，允许从对GnRH神经元的同步录音。

Watch this Scientific Journal Video about Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices at JoVE.com

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

在神经系统的活动往往需要在特定人群中的神经元的同步行动，从潜在的排放。例如，脉冲促性腺激素释放激素（GnRH）可能需要GnRH神经元之间的协调活动。我们提出我们的方法途径，可靠取得的弥漫性分布的GnRH神经元的同步电生理记录。

双体录音从促性腺激素释放激素（GnRH）神经元下丘脑薄片鉴定绿色荧光蛋白（GFP）

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

dual-somatic-recordings-from-gonadotropin-releasing-hormone-gnrh

Research

JoVE Journal

Neuroscience

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

22.2K 次观看.  University of Texas San Antonio - UTSA. 在神经系统的活动往往需要在特定人群中的神经元的同步行动，从潜在的排放。例如，脉冲促性腺激素释放激素（GnRH）可能需要GnRH神经元之间的协调活动。我们提出我们的方法途径，可靠取得的弥漫性分布的GnRH神经元的同步电生理记录。

视频: Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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 ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

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 ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...