由美国国家科学基金会（0854551）资助。

1。实验前的准备工作<ol><li>准备以下的解决方案（见表1的组合物）：（一）NFR1升（普通青蛙林格氏），（二），100毫升10％的盐水，（三）100毫升CMF（Ca 2 +的2 +的溶液/镁）和（d）1毫升ITS（胰岛素 - 转铁蛋白 - 硒，Sigma公司I1884）和（e）100毫升的L-15培养基和（f）10毫升HCG（人绒毛膜促性腺激素西格玛CG-10），（克）100毫升K +-内部溶液和（h）100毫升K +-内部溶液为两性霉素B溶液1的渗透强度应进行检查，以确保它是约260毫渗使用的蒸气压渗透压计（ 例如 WESCOR模型＃5100C） 。 </li><li>过滤消毒的解决方案（b）和（c）所示。解决方案（一）（b）和（c）可以被存储长达三个月在4℃下准备药瓶含有冻干的粉末通过25号注射器针头和注射器过滤器通过加入1毫升去离子水H 2 O的溶液（d）。这最后的溶液c可在4℃下存储30天。 </li><li>准备溶液（e）在层流罩，通过组合所有的组件在一个烧杯或烧瓶。过滤消毒的最终解决方案，并转移到无菌的离心管10毫升等分。贮存于-20℃下</li><li>准备的HCG（溶液（f）段），加入10毫升去离子水H 2 O药瓶含有冻干的粉末通过25号注射器针头和注射器过滤器。此最终的解决方案可以在4℃下存储30天。 </li><li>制作至少两个显微切割工具通过胶合一个Minutien引脚（26002-10，精细科学工具）与氰基丙烯酸酯胶的玻璃巴斯德吸管的端。允许引脚的锋利的末端延伸过去的吸液管的端部约0.5厘米。 </li><li>前两天准备的文化，诱导育种以下步骤。通过观察泄殖腔突出，红色的女性和黑暗的色素沉着的平面苏识别的滋生准备对非洲爪蟾制品表面的前爪的男性。的时间，净重青蛙和保持它的腹侧面的净水槽，因此，它不能逃脱。通过网和皮下背淋巴囊注射1毫升溶液（F）。 </li><li>将养殖对在有盖的水10加仑的油箱。为了确保动物不会肆意践踏新铺设和受精的鸡蛋，安装的屏蔽层与网目尺寸约1.5“安装约1-2英寸以上的罐底（ 图1A）。 </li><li>不受干扰12-48小时，直到离开青蛙的动物是在抱，受精卵观察屏幕下方的地板上（ 图1B）。 </li><li>从油箱中取出的动物，但静置至少24小时不再离开的鸡蛋。六个星期后，这对青蛙可以重新孕育。 </li><li>松开胚胎从罐底，然后将其转移到四，五60x15毫米崇拜URE菜肴，含有10％食盐水（溶液b）。根据计划Niewkoop和Faber（见20），排序的胚胎阶段。有用的胚胎将是那些在阶段22-24。 图2A示出了在约级22胚胎太多的发展是有用的（约级28），而图2B示出的胚胎。重要的是要选择健康的胚胎，在外观上与浅棕色和白色的斑点是光滑的理想。胚胎有大的黑色或白色斑块，一般都是不健康的，无法使用。 </li></ol> 2。 非洲爪蟾胚胎的显微切割<ol><li>层流罩，标签内，并，填补约一半60x15毫米无菌培养皿用10％盐水;和与CMF之一。 </li><li>使用无菌，玻璃巴斯德移液管转移五至十个阶段22-24的胚胎成一个含有10％食盐水的菜肴。援助的立体声变焦解剖显微镜油烟机内（0.6〜5倍与10倍的目镜），取出的果冻大衣，卵黄膜从每个胚胎，使用两对不育＃5镊子（11251-30，精细的科学工具）。 （请参阅图3A和3B）。 </li><li>通过他们的时间，通过剩余的10％的盐水的两道菜，最后放入菜含有CMF清洗裸露的胚胎。使用新的，为每个传输和无菌移液管从培养皿转移到培养皿的溶液的体积减至最小。 </li><li>反过来，轻轻地但坚定持有每个胚胎与对钳子，并使用显微切割工具，塑造在步骤1.5中，移除位于最背侧的方面的动物的神经管和相关的肌节。做到这一点，通过使三片，在任一端的位置的神经管和三分之一只是腹侧（ 图4A）中的一个。将的每个解剖神经管/肌节的菜到一个干净的部分，从蛋黄造粒es和其他碎片（ 图4B）。背腹轴和神经管和肌节的位置， 如图4中所示。 </li><li>经过约15分钟，在这个解决方案（CMF）使用镊子，解除皮肤色素沉着的解剖组织，并丢弃。一个额外的30-60分钟后，将细胞将形成“砂桩”，因为它们变得脱离彼此（ 图4C）。 </li></ol> 3。制备神经肌肉文化的<ol><li>解冻的10毫升管的培养基和无菌条件下添加70微升ITS和35纳克/毫升，脑源性神经营养因子（Sigma公司B3795）。 </li><li>标签和填写无菌的35 mm培养皿（世界精密仪器FD35-100）约一半的L-15培养基（解决方案（E）），每个胚胎解剖。 </li><li>制作镀敷玻璃巴斯德吸管的每一端，而锥形部分保持在一个火焰通过抓住移液器。拉出的端部除了至约10厘米，然后折断端产生约0.2毫米的尖端。 </li><li>使用电镀移液器吸“砂桩”来自一个胚胎的最小化的溶液的量绘制。上驱逐细胞培养皿的底部。板的细胞分成几行。观察镀敷未分化的细胞（ 图5A和图5B）。 </li><li>发表镀细胞，静置至少15分钟的菜肴，让细胞附着的菜。 </li></ol> 4。膜片钳神经肌肉突触<ol><li>镀细胞培养12-24小时后，采取不同的形态特征：肌肉细胞变得纺锤状和脊髓神经元延伸很长的过程（ 图6）。可以确认，同时对电生理记录功能的突触神经轴突膨体和肌肉细胞之间的联系。 “M”，“S”和“V”分别指的平滑肌细胞，神经元胞体和突触前静脉曲张。 </li><li>准备2块电极记录：突触前静脉曲张的肌肉细胞。 </li><li>对于突触前电极，准备一个解决方案如下：两性霉素含含5毫克两性霉素B（适马A4888）至1.5 mL离心管中加入100μlDMSO。涡此溶液中10秒。接下来，添加10微升该溶液的第二微量离心管中，其中包含625微升ķ两性霉素B（溶液（小时））+内部 ​​溶液。涡如上。 </li><li>填充的突触前电极与两性霉素-含有K +步骤4.3中制备的内部溶液与K +-内部溶液（溶液（g））和突触后电极。 </li><li>更换介质与NFR在培养皿中，并重新将其配有相衬光学倒置显微镜。 </li><li>两个电极定位高于各自的目标。获取全细胞合作nfiguration与肌细胞的前穿孔修补程序配置的静脉曲张电极的电极。 </li><li>以确认功能的突触，去极化静脉曲张膜片钳放大器（ 例如 Axopatch 200B中，Molecular Devices公司）在软件的控制下（ 例如，通过膜片钳pClamp 9，Molecular Devices公司）和观察的同时突触前和突触后电流。 图7示出了电流在突触前静脉曲张，以10 mV递增，从-30毫伏至+40 mV的去极化电压的步骤。在突触前细胞观察的内向电流进行由Na +和Ca 2 +和K +的外向电流。电流在突触后细胞是静脉曲张的神经递质释放的反应，主要是由Na +通过烟碱型乙酰胆碱受体通道。 </li></ol>

<ol>
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没有利益冲突的声明。

成功的运动神经元和肌肉细胞共培养的关键步骤是使用适当的上演了从非洲爪蛙的人工繁殖胚胎的未分化脊柱神经细胞和成肌细胞，并仔细无菌解剖。受精卵应保持原状，直到他们达到约10级左右移动他们更快停止他们的发展。健康的胚胎，确定一个平滑的外观和棕色和白色斑驳的色素。第一阶段22日至24日的胚胎是最有用的，因为这是点后神经管闭合之前，在开发过程中的心肌细胞已分化显着。此外，从旧的胚胎细胞未能，在CMF分离。护理时，应采取删除，因为它牢固地粘附在胚胎的卵黄膜。被撕裂膜应使用锋利的镊子，使胚胎出现完整的。另一个重要的precaution是小心地将细胞在培养皿的底部（而不是让他们在那里定居）。这是首选方法，因为这增加的可能性，这些细胞将坚持培养皿中。 功能神经传导神经和肌肉之间可以就在突触后记录，并常可观察自发神经支配肌肉收缩后确定。联合国神经支配的肌肉细胞的不抽搐文化的。突触后记录单是有用的小终板电流或电位，但诱发发布的测量需要突触前和突触后电流的刺激，以及相关的需要双膜片钳记录。 这里描述的除了配对补丁的记录方法，该制剂提供了机会，介绍第三吸管在神经元的胞 ​​体5，这使得动作电位的产生THAt可以传播到突触前的静脉曲张，导致神经递质的释放。此外，假定，被认为是介导或调节突触传递的代理可以被引入到两侧的突触：通过肌肉细胞移液管或通过扩散从三分之一的移液管，放置在苏摩。

<table border="1"><tbody><tr><td> 设备/用品 </td><td> 供应商 </td><td> 目录/型号 </td><td></td></tr><tr><td>胰岛素 - 转铁蛋白 - 硒</td><td>西格玛</td><td> I1884 </td><td></td></tr><tr><td>人绒毛膜促性腺激素</td><td>西格玛</td><td> CG-10 </td><td></td></tr><tr><td>蒸气压渗透压仪</td><td> WESCOR </td><td> 5100C </td><td></td></tr><tr><td> Minutien销</td><td>精细科学工具</td><td> 26002-10 </td><td></td></tr><tr><td>两性霉素B </td><td>西格玛</td><td> A4888 </td><td></td></tr><tr><td>镊子</td><td>精细科学工具</td><td> 11251-30 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 解决方案名称 </td><td> 食谱 </td></tr><tr><td> （一）NFR（普通青蛙林格氏液（以mM计） </td><td> 116 2氯化钠，氯化钾，1.8的CaCl 2，5的Na +-HEPES，pH值7.35。 </td></tr><tr><td> （二）10％的食盐水</td><td> 10 ML NFR90毫升去离子H 2 O </td></tr><tr><td> （三）CMF（的Ca 2 + / Mg 2 +的免费的解决方案） </td><td> 2 125氯化钠，氯化钾，1.2 EDTA，5的Na +-HEPES，pH值为7.35。 </td></tr><tr><td> （五）L-15培养基</td><td> 50毫升L-15媒体（适马L1518）+ 50毫升NFR。 </td></tr><tr><td> （克）K +-内部溶液（以mM） </td><td> 116氯化钾，1的NaCl的MgCl 2，1，10 EGTA，5的HEPES，pH 7.3的</td></tr><tr><td> （高）K +-两性霉素B的内部溶液（以mM） </td><td> 52 K 2 SO 4，38氯化钾，EGTA，5的HEPES，pH 7.3的</td></tr></tbody></table></td></tr></tbody></table> TABLE 1。

simultaneous pre- and post-synaptic electrophysiological recording from xenopus nerve-muscle co-cultures

已取得的无脊椎动物的准备工作，最显着的鱿鱼巨大的突触的突触前神经递质释放的离子电流的耦合多的信息。然而，除了用于制备这里描述的，存在几脊椎动物制剂，它能够使神经递质释放和突触前的离子电流的同时测量。胚胎爪蟾运动神经元和肌肉细胞中，可以在简单的培养基在室温下一起成长，它们将形成功能性突触在1224小时内，并且可以被用来研究数天的神经和肌肉细胞的发育和突触的相互作用（直到过度生长）。这些共培养超过其他脊椎动物制剂的一些优点包括简单的准备，保持文化和在室温下工作的能力，和突触形成2就绪无障碍-4。制品被广泛地用于研究的生物物理特性的离子通道及突触前递质释放的调节5-8。此外，准备借给其他用途，包括研究神经轴突生长和突触9-12，神经递质的释放13日至15日 ，在神经调节16,17扩散使者的作用的分子机制，并在体外突触可塑性18 - 19。

图4B显示了一个孤立的脊髓/肌节立即移走后，从22 爪蟾胚胎。 图4C示出的阶段后，在Ca 2 +-Mg系2 +的溶液的皮肤去除和孵化（CMF，溶液（背视图c）条），细胞离解成一个“砂桩”，并准备好用于电镀。电镀后立即很少表现为培养基细胞形态变化（ 图5B），但不同的形状后二十四小时文化。肌肉细胞变成纺锤状，而神经元保持球形，而延伸的神经突起，与肌肉细胞突触的精心varicosites在（ 图6）。同时从突触前的静脉曲张和突触后肌细胞（ 图7）显示配对的膜片钳记录突触前抵港及离港的电流与神经递质的释放和resultaNT兴奋性突触后电流。 <img src="/files/ftp_upload/50253/50253fig1.jpg" alt="图1"/> 图1：A：繁殖对的青蛙交配屏幕桶之上。B：青蛙抱受精卵。 <img src="/files/ftp_upload/50253/50253fig2.jpg" alt="图2"/> 图2。答：“适合”的阶段，22胚B：“不适合”第28期胚胎。比例尺为1毫米。 <img src="/files/ftp_upload/50253/50253fig3.jpg" alt="图3"/> 图3：A：第一阶段前22胚胎卵黄膜和透明带的去除。箭头指向果冻外套的外边缘。卵黄膜紧贴的电磁制动哟，几乎是无形的。B：裸露的胚胎。比例尺为1毫米。 <img src="/files/ftp_upload/50253/50253fig4.jpg" alt="图4"/> 图4 A：22胚胎阶段用虚线表示被解剖的部分。B：急性分离的胚胎背侧部，“D”和“V”是指背部和腹部方面的胚胎，而“M”和“n”表示的mytomes和神经管的大约位置。C：“沙桩”的细胞60分钟后，在CMF。比例尺为 A，表示1毫米和0.5毫米，B和 C。 <img src="/files/ftp_upload/50253/50253fig5.jpg" alt="图5"/> 图5。A：细胞在培养后，立即电镀电源鉴于5倍。 B：40倍相同的文化。规模酒吧A，代表40微米和5微米B. <img src="/files/ftp_upload/50253/50253fig6.jpg" alt="图6"/> 图6。识别的神经元胞体（S），突触前的静脉曲张（Ⅴ），和突触后的平滑肌细胞（M），在培养的神经肌肉接合。比例尺：5微米。 <img src="/files/ftp_upload/50253/50253fig7.jpg" alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/50253/50253fig7highres.jpg"/> 图7。突触前（顶部）和突触后电流（底部）见于响应于应用10 mV的电压增量步骤提交到从-30毫伏至40毫伏的突触前的静脉曲张。电压的步骤，表示旁边一些的突触前的痕迹。这两种细胞的潜力-70毫伏。

Watch this Scientific Journal Video about Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures at JoVE.com

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

本视频演示了用来种植原代培养的胚胎的程序<em&gt;爪蟾</em&gt;神经和肌肉细胞和该制剂的同时前和突触后的膜片钳记录的用处。

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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Research

JoVE Journal

Neuroscience

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

10.8K 次观看.  David Geffen School of Medicine at UCLA. 本视频演示了用来种植原代培养的胚胎的程序爪蟾神经和肌肉细胞和该制剂的同时前和突触后的膜片钳记录的用处。

视频: Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

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 ...

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 ...

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 ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...