这项工作是由来自美国国立卫生研究院（F32 NS059120到RLM和R01NS25217和P30NS048154到ABR）的资助。

作者宣称没有竞争的经济利益。

这里描述的方法允许脊髓的最小解剖后斑马鱼胚胎的感觉和运动神经元的电和形态学特征。神经元至少1小时，强加给这些录音时间限制保持健康。神经元已经使用标准的全细胞构型记录，以及从核贴剂;后者的方法最小化，可以排除离子电流9的详细研究生物物理空间钳的问题。 一个重要的挑战是实现胚胎或幼虫室的坚定执着，以去除皮肤和执行提供访问感?...

<html>乔治·斯特雷伊辛格率先使用斑马鱼 ，俗称斑马鱼作为脊椎动物发育10的遗传分析模型系统。该模型提供了几个优点，包括:（1）相对简单和便宜畜牧业; （2）外部受精，从而允许方便地从最早的发育阶段的胚胎;和（3）的透明胚胎，从而允许细胞，组织和器官的直接和重复观察，因为它们形成。 在随后的几十年里，进行了若干改进进一步增加了斑马鱼模型的力量。特别是，正向遗传学屏幕和全基因组测序工作发挥了重要作用的基因突变和基因关键的标识许多发育过程11，12，13，14，"&gt; 15，16。Gateway克隆方法已经允许转基因的常规应用方法17,18。在基因组编辑的最新进展，通过转录激活状（TALEN的）中列举和群集规则间隔开的短回文重复序列（CRISPR）-Cas9核酸酶，允许有针对性地引入突变，以及敲除和敲入接近19，20，21，22，组合，这些方法使斑马鱼为底层的具体行为和一些人类疾病的遗传机制的研究提供强大的模型23，24，25，26，27。 今年工作重点放在发展心理调节和电活动的神经发育中的作用。重点是脊髓，为此，斑马鱼模型提供了几个优点。首先，它是相对容易的，在胚胎和幼体阶段访问斑马鱼;因此，可以在具有较少的神经元和较简单的电路28，29的发育阶段研究脊髓功能。此外，斑马鱼脊髓具有一组不同的神经元，类似于其他脊椎动物中，因子30，31，32，33，34，35所证明由转录的特性和特征图案。 旨在揭示背后脊髓电路的功能的机制的多数在斑马鱼的研究中，尤其是支持运动的，是可以理解集中于幼虫阶段36，37，38，39，40，41，42，43。然而，许多形成脊柱机车网络中的神经元的启动它们的分化在早期胚胎阶段，〜9-10小时后受精（HPF）44，45，46，47，48，49，50，51。鉴于此，了解如何脊髓神经元的形态学和电学性质发生和胚胎和幼虫阶段之间的变化是一个重要overa的运动电路形成和功能LL理解。 这里描述的解剖方法允许从脊髓神经元膜片钳记录，并在胚胎阶段（〜17-48 HPF）和幼体阶段（〜3-7天受精后[DPF]）得到成功应用。该方法限制了解剖，以提供对感兴趣的神经元所需要的量。所述协议从多数的其它公开的方法的不同之从在兽医缝合胶斑马鱼脊髓神经元的记录使用的，而不是一个细钨针，胚胎或幼虫附着到记录室。的两种不同的方法的可用性（ 即，缝合胶相对于钨针）用于安装所述斑马鱼的胚胎或幼虫用于电生理分析为研究人员提供替代方案，以实现其特定的实验目标。 首先，从弹出访问和记录过程<html>初级感觉神经元的ulation，Rohon-胡子细胞，进行了描述。这些神经元的细胞体位于背侧脊髓内。 Rohon-胡子细胞存在于许多脊椎动物物种，在开发早期分化，和背后胚胎触摸响应6，44，47，48。 第二，用于访问和从脊髓运动神经元的记录程序的详细。期间神经发生的两个波斑马鱼脊髓运动神经元引起的。在出生较早初级运动神经元出现在原肠胚形成（〜9-16 HPF）的端部，只有3-4本每hemisegment 45,46，49初级运动神经元。相反，次级运动神经元的后出生人口是比较多，而且在长时间内产生，起始于14〜HPFEF"&gt; 45,50。在中间躯干段二级运动神经元起源主要是由51 HPF 50完成。二级运动神经元都被认为是运动神经元的羊膜对方46。有趣的是，脊椎神经元，经由多巴胺，调节运动在胚胎的幼虫和次级运动神经元起源和年轻幼虫50,51。初级和次级的运动神经元的每一个包括若干不同的亚型。每个初级运动神经元亚型的项目的外围轴突其支配的特性的肌肉群，从而导致刻板，识别轴突轨迹。通常，次级运动神经元遵循先前由初级运动神经元建立的轴索途径。因此，相对于轴突轨迹，原发性和继发性运动神经元是相似的，不同之处在于厚度轴突胞体和尺寸的重新更大初级运动神经元45。 三，讨论从几个类型的interneurons的记录方法。然而，在这些情况下，需要去除其它脊髓细胞的有限量的，因此，脊髓比从Rohon-胡子细胞或运动神经元的录音少完好无损。

<table><tbody><tr><td>Vacuum filter/Storage bottle, 0.22 mm pore</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>Syringe filter 0.2 mm</td><td>Whatman</td><td>6780-2502</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td>Ethyl 3-aminobenzoate methanesulfonate salt&amp;nbsp;</td></tr><tr><td>&amp;alpha;-bugarotoxin</td><td>Tocris</td><td>11032-79-4</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Tocris</td><td>4368-28-9</td><td></td></tr><tr><td>Alexa-549 hydrazine salt</td><td>Molecular Probes</td><td>A-10438</td><td>fluorescent dye</td></tr><tr><td>Spin-X centrifuge tube filter</td><td>Corning</td><td>8161</td><td></td></tr><tr><td>Glass microscope slide</td><td>Fisher</td><td>12-550C</td><td></td></tr><tr><td>Sylgard silicone elastomer kit</td><td>Dow Corning</td><td>184</td><td>silicone elastomer</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0038</td><td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td></tr><tr><td>Borosilicate glass capillaries</td><td>Drummond Scientific</td><td>1-000-1000-100</td><td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td></tr><tr><td>Miniature barbed polypropylene fitting&amp;nbsp;</td><td>Cole-Palmer</td><td>6365-90</td><td></td></tr><tr><td>Vetbond tissue adhesive&amp;nbsp;</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instruments</td><td>&amp;nbsp;Model P-97&amp;nbsp;</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200B</td><td></td></tr><tr><td>Head stage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Motorized micromanipulator&amp;nbsp;&amp;nbsp;</td><td>Sutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Tygon tubing</td><td>Fisher</td><td>14-169-1B</td><td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td></tr><tr><td>Electrode holder</td><td>Molecular Devices</td><td>1-HC-U</td><td></td></tr><tr><td>Pharmaseal Three-Way Stopcocks&amp;nbsp;</td><td>Baxter</td><td>K75</td><td></td></tr><tr><td>Digitizer</td><td>Axon Instruments</td><td>Digidata 1440A</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Zeiss</td><td>Axioskop2 FS plus</td><td></td></tr><tr><td>40X/0.80W Achroplan objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Data acquisition and analysis software&amp;nbsp;</td><td>Axon Instruments</td><td></td><td>PClamp 10 - Clampex and Clampfit&amp;nbsp;</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>Model P-97</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection and Recording Solutions (in mM)&lt;/strong&gt;</td></tr><tr><td>All solutions, except the intracellular, are stable for ~2-3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td><td></td><td></td><td></td></tr><tr><td>The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20 &amp;deg;C) in small aliquots that are individually thawed on the day of use.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dissection/Ringer&amp;rsquo;s solution</td><td></td><td></td><td>145 NaCl, 3 KCl, 1.8 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 10 HEPES; pH 7.4 (with NaOH)</td></tr><tr><td>Pipette (intracellular) recording solution</td><td></td><td></td><td>135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td></tr><tr><td>Bath (extracellular) recording solution/voltage and current-clamp</td><td></td><td></td><td>125 NaCl, 2 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 5 HEPES; pH 7.4 (with NaOH).</td></tr><tr><td>Alexa-594 hydrazine salt stock solution.&amp;nbsp;</td><td></td><td></td><td>Prepare a 13.2 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O, aliquot (~100 &amp;micro;l) and store at -20 &amp;deg;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&amp;nbsp; with a centrifuge tube filter.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immobilizing agents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.4% ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td></td><td></td><td>Prepare a 0.4% stock solution in 0.2 M Tris, pH 9 (0.4 g Tricaine/100 mL 0.2 M Tris</td></tr><tr><td colspan="4">Adjust pH to 7 with NaOH and store at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute the stock solution ~25 fold in embryo media</td></tr><tr><td>250 mM &amp;alpha;-bungarotoxin</td><td></td><td></td><td>Prepare a 250 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O (1 mg/500 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td></tr><tr><td>1 mM Tetrodotoxin</td><td></td><td></td><td>Prepare a 1 mM stock in ddH2O (1 mg/3 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td></tr></tbody></table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

斑马鱼，首先介绍了作为一个发展模式，在许多领域都得到了普及。饲养迅速发展生物体的大数自如，与胚胎光学透明度结合，担任该模型的初始吸引力属性。在过去的二十年里，这种模式的成功得到了进一步的顺从大规模诱变筛选，并通过易于转基因的推动。最近，基因编辑方法扩展了模型的力量。 对于神经发育研究，斑马鱼胚胎和幼虫提供到可应用多种方法的模型。在这里，我们专注于让神经元，电兴奋性的本质属性的研究方法。我们对斑马鱼的脊髓神经元的电生理研究编制涉及使用兽医缝合的胶水，以确保准备录音室。记录替代方法从斑马鱼胚胎和幼虫涉及使用细钨针1，2，3，4，5的制备腔室的连接。钨销是最经常用于安装在横向取向的制备中，虽然它已被用于安装幼虫背的一面朝上4。缝合胶水已被用于安装在两个方向的胚胎和幼虫。使用胶水，一个最小的夹层可以执行，允许访问脊髓神经元而不使用酶处理的，从而避免了任何所得的损坏。然而，对于幼虫，有必要施加一个简短酶处理以除去脊髓周围的肌肉组织。这里所描述的方法已被用来在几个developmenta研究运动神经元的interneurons和感觉神经元的内在电性能升级6，7，8，9。

（ 图5A和5B），我们已经成功地从Rohon胡子神经元记录在17个HPF胚胎到7 DPF幼虫。当Rohon-胡子细胞记录，将制备物固定背的一面朝上。这种安装允许基于它们的表面背的位置和大胞体大小Rohon-胡子细胞的明确识别。识别另外由这些神经元的超极化的定型静息膜电位（ 图5， 小图表 ）6，54</su...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

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

这手稿描述了从斑马鱼胚胎和幼虫的脊髓神经元的电生理记录的方法。制备维持神经元在原地 ，往往涉及到最小的清扫。这些方法允许对多种脊髓神经元的电生理学研究中，从通过早期幼体阶段的初始电兴奋采集。

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 ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Research

JoVE Journal

Neuroscience

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

11.4K 次观看.  University of Colorado Anschutz Medical Campus (UCAMC). 这手稿描述了从斑马鱼胚胎和幼虫的脊髓神经元的电生理记录的方法。制备维持神经元在原地 ，往往涉及到最小的清扫。这些方法允许对多种脊髓神经元的电生理学研究中，从通过早期幼体阶段的初始电兴奋采集。 

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

发育生物学

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

医学

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

Source: Benedetti, L., et al. <a href="https://app.jove.com/v/55297">Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates a non-invasive assay to study electrophysiology of motor neurons in zebrafish embryos. Upon measuring tail coiling in the embryos as a result of spontaneous depolarization of the spinal cord motor neurons, a sodium channel blocker is applied to reduce the coiling frequency. Next, the embryos are immobilized in agarose and placed under a fluorescence microscope. The genetically engineered motor neurons express a voltage-sensing biosensor with a donor and an acceptor fluorophore. A decrease in acceptor fluorophore emission indicates a reduction in the spontaneous depolarization of motor neurons due to the sodium channel blocker.

一种研究斑马鱼胚胎中运动神经元电生理学的无创方法

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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斑马鱼

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