作者想，感谢他们及早建立斑马鱼在实验室的彼得·卡斯特罗和马修Dinday。这项工作是由美国国立卫生EUREKA（＃R01NS079214-01）资助。

1。鸡蛋的生产和收集<ol><li>斑马鱼的饲养标准程序，前面描述的8。简单地说，成年斑马鱼在养殖箱分频器的地方。第二天早上，在房间里的灯都分频器从坦克和鱼类育种允许不受干扰的交配时间约20至60分钟。 </li><li>从繁殖罐的鸡蛋被收集在过滤器并和用鸡蛋水漂洗。卵，然后转移到陪替氏培养皿用鸡蛋水。用移液管未受精的卵子和碎片被删除。 </li><li>将培养皿包含收集鸡蛋的培养箱中（28-32℃）。卵孵化后，大约2天受精后（DPF），去除绒毛和任何其他的碎片，用移液管。 </li><li>所需的天受精后（3〜8 DPF）的培养皿中自由游动的幼虫和实验室的长椅上，在室温下删除。 </li></ol><ol><li>用微拉马，拉1.2毫米外径硼硅玻璃毛细管橡皮泥斜坡铺设超过两针，并存储在一个150毫米的培养皿或空吸管盒。针可以预先拉出。取决于可用放大器探头类型，可以使用不同OD玻璃毛细管。 </li><li>回程微电极与细胞外记录用的Nalgene针筒式过滤器（4毫米）和Microfil长丝（华纳精密仪器）连接用1ml注射器的溶液（2 M氯化钠）。摇动或敲击丸向针尖，直到有很少或根本没有气泡，剩余的。 </li></ol> 3。琼脂中的固定化<ol><li>准备一个新鲜的溶液中1.2％低熔点琼脂在蛋水。将琼脂溶液在约37℃下在水浴中集</li><li>准备盖玻片，在录音室和冷冻室（5-10分钟）。录音室MATC为h的电生理钻机上使用。我们使用一个低调的开放式钻石浴成像室，从华纳仪器（RC-26GLP型）。 </li><li>使用巴斯德移液管，在一个干净的培养皿中板的小液滴的蛋水的地方之一幼虫的斑马鱼。这种液滴，添加含有麻醉剂（0.02％三卡因）和瘫痪剂（1毫克/毫升α-银环蛇毒素）的溶液的液滴。 </li><li>监控斑马鱼的运动的损失（5〜10分钟）。 </li><li>从冰箱中取出盖玻片/录音室。将腔体视显微镜的阶段。使用移液管，混合液滴和传输解决方案（鱼）的盖玻片/录音室几毫升1.2％的低熔点琼脂糖。 </li><li>使用移液管尖的或细的钝针，在体视显微镜下，使背侧方面的鱼是暴露于琼脂糖凝胶表面的位置幼虫。允许琼脂糖硬化（5-10分钟），然后使用平面的锅铲传输琼脂b锁定到录音室成立于电钻机。 </li></ol> 3。外场录制<ol><li>添加2-5毫升斑马鱼的记录介质的录音室。如果药物的改变是必要的，在约1毫升/分钟的速率与记录溶液的灌注腔室。无氧化的录音解决方案是必要的。 </li><li>将微小电极（尖端直径约为1微米，2-7MΩ）到放大器探头安装在一个三维的微操纵器。检查，显微操作器是在一个适当的位置，以允许的范围内的移动和调整。把微电极尖端平面的显微镜，走下舞台，和使用下一个点正上方和前面的斑马鱼的头稍微粗调。 </li><li>随着"电流钳"模式零的电极中的放大器。 </li><li>使用较低的粗调，使电极尖刚好接触的Surface的斑马鱼，稍前方的前脑。 </li><li>使用精细调整步骤推进电极头，直到它穿刺皮肤的斑马鱼。允许沉降，然后提前几微米到前脑的电极。 </li><li>记录电活动在电流钳模式使用Axoscope软件的。采集数据的采样速率为10 kHz。 </li></ol>

<ol>
	<li>Clark, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

没有利益冲突的声明。

这里介绍的方法使细胞外记录大脑活动的一个非常敏感和快速的分析。这些录音是类似的脑电图（EEG）监测常用的评估存在的异常放电（ 即癫痫发作）在啮齿类动物模型癫痫11和患者12。细胞外记录可以结合药理的操作，如下所示。也可用于这些类型的记录，以评估潜在的癫痫表型的转基因斑马鱼。确定，在ENU的突变屏幕（见斑马鱼资源中心， <a href="http://zebrafish.org/zirc/home...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>琼脂糖低熔点</td><td>费舍尔的科学</td><td> BP1360-100 </td><td>溶解于1.2％的胚胎媒体</td></tr><tr><td>记录媒体</td><td>费舍尔的科学</td><td> BP3581，P330-3，BP410，BP214-500，D16-1，C77-500 </td><td> 1毫摩尔NaCl，2.9 mM KCl中，10mM的HEPES，1.2毫摩尔MgCl 2，10mM的葡萄糖，2.1 mM的CaCl 2的 pH值至约7.3，用1N NaOH </td></tr><tr><td>三卡因</td><td>银色实验室</td><td> MS-222 </td><td> 0.02％ </td></tr><tr><td> α-银环蛇毒素</td><td> TOCRIS生命科学</td><td> 2133 </td><td> 1毫克/毫升</td></tr><tr><td>毛细玻璃管</td><td>华纳仪器</td><td> G120TF -3 </td><td>拉出到2 -7MΩ的电阻</td></tr><tr><td>膜片钳放大器</td><td>华纳仪器</td><td> PC-505B </td><td>使用华纳放大器的电流钳模式;在2 mV / PA和贝塞尔滤波器在2K的增益设置。同类机型可根据制造商的说明。 </td></tr><tr><td>过滤器/放大器</td><td>天鹅座技术</td><td> FLA-01 </td><td>我们使用一个新天鹅前置放大器增益设置为10〜20;截止频率设置为1-2K;陷波滤波器。同类机型可根据制造商的说明。 </td></tr><tr><td>轴突A / D板和Axoscope的软件</td><td> Molecular Devices公司</td><td>轴突Digidata 1320A 8.2 Axoscope </td><td>数据被收集在Axoscope采用无间隙采集模式，在10 kHz采样。比较模型和程序可以根据制造商的说明使用。 </td></tr><tr><td>蛋水</td><td&gt;即时海洋</td><td></td><td> 3克即溶海洋海盐，2毫升10毫升去离子水，0.1％的亚甲基蓝</td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

癫痫会影响近3万人在美国和世界各地多达50万人。定义为发生自发的无端发作，癫痫，可以获取到大脑或基因突变的结果的一种侮辱。在动物模型癫痫发作的努力，主要用于收购侮辱（惊厥药，刺激或脑损伤）和遗传操作（反义击倒，同源重组或转基因）在啮齿类动物。斑马鱼是脊椎动物模型系统1-3，可以提供一个有价值的替代老鼠癫痫研究。斑马鱼被广泛应用于脊椎动物的遗传学研究或发展，对哺乳动物的遗传相似性，表现出高度表达的同系物〜85％的已知的人类单基因遗传性癫痫突变。由于它们的小尺寸（长4-6毫米），斑马鱼幼虫可以保持低的流体体积为100微升，在早期的发展和阿拉进行期间，在多井板。试剂可直接加入到该溶液中，在胚胎发育，简化给药，从而实现快速的在体内筛选试验化合物4。可用于合成寡核苷酸（吗啉代），致突变，锌指核酸酶和转基因方法，以迅速产生基因敲除或突变的斑马鱼5-7。这些属性提供了前所未有的斑马鱼研究的统计动力分析优势的研究啮齿动物的神经系统疾病，如癫痫。 ，因为癫痫研究的"金标准"是描述监测和分析的异常放电起源于一个中央的大脑结构（ 即癫痫发作），斑马鱼幼虫的大脑活动的方法，有效地记录在这里。该方法是一种适应传统的细胞外记录技术，并允许稳定的长期监测在完整的斑马鱼幼虫的大脑活动。小号充足的记录上所显示的急性发作引起的惊厥药和自发发作在转基因鱼浴应用。

在图1中所示的实施例的电子照相记录的前脑琼脂嵌入的斑马鱼幼虫的癫痫样放电。在这些样本中的大振幅的多穗型脉冲放电引起的惊厥药浴应用，40毫米毛果芸香碱（A; 6 DPF）或1毫米印防己毒素（B; 8 DPF）。在这些记录中，固定，和琼脂嵌入式斑马鱼的连续监测90分钟。鱼这些记录的条件下继续生存24小时。药物添加到泳季介质和通常扩散到琼脂在30至45分钟之内引出在幼虫斑马鱼前脑?...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

外场电位记录在幼虫斑马鱼的前脑的一个简单的方法来描述。该方法提供了一个强大的<em在体内</em&gt;读出癫痫样活动。这种技术可以使用转基因斑马鱼幼虫携带癫痫相关基因或癫痫发作诱发的惊厥药给药。

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 ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

JoVE Journal

Neuroscience

17.0K 次观看.  University of California, San Francisco. 外场电位记录在幼虫斑马鱼的前脑的一个简单的方法来描述。该方法提供了一个强大的

视频: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

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 ...

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 ...

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 ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

对于系统电化学神经记录电极和电生理评价方法

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

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 ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

电生理记录<em&gt;果蝇</em&gt; Trichoid Sensilla响应低挥发性气味

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...