我感谢大号渡边，男Gorczyca和有益的意见和讨论的吉原实验室的其他成员。我感谢K.斯科特和Looger研究苍蝇股票，横山的实验表明，伸井口的技术帮助，和信子吉原物料信息。这项工作得到了研究所，心理健康教育资助MH85958和我的伍斯特基金会

1。构建苍蝇<ol><li>塑造一个平台，从35毫米猎鹰盘的盖子融化侧壁和雕刻，形成一个适当的角度和厚度（ 图1a，图2）。钻一个洞，在这个平台上接受苍蝇头（ 图1A，B），这样的口器自由地接触到外面室（ 图1a）。削减的Pipetman尖底，与大小匹配飞待观察。胶尖在图1a所示的平台完成的苍蝇（ 图2）。 </li></ol> 2。准备观察粉煤灰， <ol><li>挨饿24小时的成年果蝇在25°C间事先放置在一个小瓶只用湿纸巾，如果饥饿是必要的实验。 </li><li>麻醉由放置在15毫升塑料管站在冰上飞。 </li><li>使用镊子，插入室的苍蝇苍蝇，轻轻一推在飞，直到它无法动弹。然后，插入一个插件保留的苍蝇。 （ 图1A，B）。 </li><li>近端长鼻（主席台）孔的内边缘密封周边配件;应用少量的光固化胶（EvoFlow Tetric）双方从主席台上方的外（ 图3中的箭头）。也适用于胶从里面的苍蝇头背侧部分和孔的内边缘（ 图1b）之间的空间。允许讲台上自由移动，应小心，以防止任何触摸主席台上，通过使用睫毛（或相媲美的东西）传播胶水胶水。最后，治愈与蓝色光固化过多的热量，以避免损坏飞足够的光照最弱的胶水。删除插件。 </li><li>一个线程持有的部分解除了讲台上（ 图3中的箭头），可以采用稳定苍蝇“避免磕碰的咽的一部分，特别一次过拨款及特别一次过拨款的腹侧部分，揭露，尤其是成像突触前末梢神经元Gr5a 8，长鼻完全缩回时咽的一部分，这是由覆盖头部。 </li><li>填补了无糖盐水含（毫米）：氯化钠，140平台的表面;氯化钾，2 4.5 氯化镁 ， 氯化钙 ，1.5和肝素钠的氢氧化钠，pH值7.1 10 5。这种无蔗糖的生理盐水，也有类似的强壮那些常用的蔗糖含salines如HL3.1 11。 </li><li>打开头壳使用削尖的秘诀，如剪刀，这是一个定制的设备和设计最初是由博士，日本，景行山冈夹角质层及气管暴露SOG（ 图4）钨刀片和镊子。头部的角质层腹侧边缘尽可能削减腹，背边割而不是作为关键。首先，通过一个切口使用钨刀片后缘。然后，穿过侧面和使用削尖钳前边缘。切角质层件应解除与镊子和删除。应触角和触角神经切断由削尖钳。 </li><li>为了避免在录音移动文物，大脑必须稳定通过清除苍蝇的某些部分。首先，有选择性地删除假手术和表皮之间的空气袋。在年底向咽部及特别一次过拨款，以清除咽部和大脑之间的连接在年底削减食道。然后拉出肌肉16 12，最后，分离气管连接大脑和角质层。 </li><li>奥林巴斯显微镜BX51WI连接到一个旋转盘共聚焦显微镜系统（Improvision在珀金埃尔默）（ 图5）上的阶段，我们建立我们的苍蝇。一个水浸泡镜片，如40X/0.8 NA，浸入浴（ 图6 </strong&gt;）。 </li></ol> 3。的Ca 2 +的脑成像<ol><li> 钙离子成像使用GCaMP3.0 1，9，玛蕊乐等建立的方法。（ 图6）。使用旋转盘共聚焦显微镜（Improvision），在4Hz的一个单一的光学部分使用491nm激发激光122ms曝光时间，重点在该地区的利益（运动神经元的细胞体或中间神经突触前末梢味觉的感觉神经元）与速度的软件，VER。 4.3（Improvision）。 </li></ol> 4。刺激的长鼻<ol><li>小灯芯从尖（ 图7）凸出（从日本和纸，见特定试剂表 ）到一个注射器针头吸100MM水蔗糖溶液。 </li><li>放电到灯芯的小水滴的蔗糖溶液，然后，吸它，立即江前ê申请浸泡和纸纸灯芯喙尖。和纸灯芯只应适用于瞬间，以避免饱食（ 图8）。监测的长鼻扩展响应（PER）的行为，使用CCD相机连接到一个解剖显微镜（ 图5），同时与Ca 2 +成像。数据必须在一小时内开始后剥离，因为每个反应不能维持一个多小时，在这些条件下。 </li></ol> 5。代表结果在主席台量角器，一双的负责解除长鼻延伸主席台的主要肌肉，运动神经元已显示强大的13甜刺激的反应。，通过GCaMP3.0 1 9 图9所示的Ca 2 +信号表示加仑4驱动，E49 13，从运动神经元的细胞体。如图所示，在视频，robuST每增加的Ca 2 +信号的同步观测。 <img alt="图1" src="/files/ftp_upload/3625/3625fig1.jpg" /> 图1。苍蝇的大脑实时成像和电生理阶段（苍蝇）的示意图。 ，苍蝇建墙的角度，允许苍蝇头走出来直。飞长鼻三段彩色编码;主席台是洋红色。孔飞用钳子插入室，从一个Pipetman尖底切下一块是插入室，以阻止苍蝇逃跑的背面，B，无聊符合苍蝇的头。余下的差距与光固化胶密封的指示，以确保没有泄漏。胶水固化后，作为一个插件Pipetman尖被删除。生理盐水倒入苍蝇等，完全覆盖大脑表面。有没有盐水泄漏，应确保ING，到年底前的苍蝇头。在B组的虚线包围的阴影区域显示部分被解剖。 <img alt="图2" src="/files/ftp_upload/3625/3625fig2.jpg" /> 图2。苍蝇的照片。胶水状的月牙墙（从胶枪）是由控制盐水灌注过程中的流动，减少使用量的生理盐水。 <img alt="图3" src="/files/ftp_upload/3625/3625fig3.jpg" /> 图3。安装在苍蝇苍蝇的正面视图。此图像显示了一只苍蝇，苍蝇如何，以及如何应用光固化胶头的苍蝇（箭头）创建生理盐水证明印章周围。如果通过飞长鼻任何盐水泄漏，则实验会失败。拴在舞台上的线程（箭头）举行的长鼻位置，所以它不是完全retrac<html>特德。否则，它会阻碍更多的腹侧部分的特别一次过拨款，并导致移动文物。 <img alt="图4" src="/files/ftp_upload/3625/3625fig4.jpg" /> 图4。与解剖苍蝇苍蝇的顶视图。此图像显示了苍蝇以下角质层去除暴露的特别一次过拨款。 16食道，肌肉和气管连接到大脑，以及选定的空气，麻袋也被删除，以防止他们扰乱大脑，从而导致钙信号文物。 <img alt="图5" src="/files/ftp_upload/3625/3625fig5.jpg" /> 图5。该仪器的监测仪器。BX51WI连接到一个旋转盘共聚焦显微镜系统（Improvision）奥林巴斯显微镜组装，边安装尼康SMZ800显微镜。此设置允许在每实时成像大脑活动的行为。 6“SRC =”/ files/ftp_upload/3625/3625fig6.jpg“/&gt; 图6。照片显示的苍蝇侧视。盐水之间夹着一层薄薄的40X水浸泡的镜头和苍蝇。 <img alt="图7" src="/files/ftp_upload/3625/3625fig7.jpg" /> 图7。示意图和纸灯芯雁皮和纸是日本传统的羊皮纸，是非常薄而光滑（灰原，日本）。它也是非常强，可以保留大量的液体。必须和纸剪成一个梯形，非常具体的尺寸，0.3毫米和0.7毫米，宽度和长度4-5毫米（ 一 ）。这梯形然后插入到了23政注射器针头尖端从0.7毫米方，也可以通过插入昆虫的脚，弯曲V形（二）稳定，C，和纸变成透明以下曝光液体，使这个实验的理想选择。 <img alt="图8" src="/files/ftp_upload/3625/3625fig8.jpg" /> 图8。天鹰，灯芯苍蝇长鼻刺激。在一个注射器针头尖端和纸灯芯一方面用操纵杆操纵即时申请。针是通过软管连接用另一只手操纵的注射器，抽吸和排放的蔗糖溶液。 <img alt="图9" src="/files/ftp_upload/3625/3625fig9.jpg" /> 图9。有代表性的成果。 ，每行为记录通过安装在立体显微镜的CCD相机。一个饥饿的苍蝇正面意见与扩展长鼻（箭头）在刺激之前的刺激，刺激后，用含有100毫米蔗糖水溶液（箭头）灯芯。照明是通过491纳米激光用于荧光，B为GCaMP蔗糖诱导GCaMP的运动神经元在主席台肌肉处于饥饿状态的量角器3.0响应。刺激前面板，底部面板后，立即刺激。比例尺，10微米，C，量化的蔗糖诱导GCaMP的3.0响应的。运动神经元响应蔗糖刺激，随后的几秒钟的恢复阶段的底线在荧光急剧增加。

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<p class="jove_content"&gt;我什么都没有透露。</p

苍蝇可同时录制的Ca 2 +信号，每行为。即使暴露于生理盐水正常每行为的大脑进行了观察。使用，而不是在原有毛细血管method7用于Kimwipe灯芯雁皮天鹰灯芯利于高度重复性和稳定的每个行为和避免了成为熟练的决策和选择一个好的Kimwipe灯芯。上述实验提示允许运动的长鼻我们成功地避免干扰，导致录音非常稳定的钙2 +信号与噪音低。有时，组织切除不充分暴露细胞或抑制运动充分，导致效果不佳。然而，一旦我们熟练，超过80％的准备工作产生了良好的效果。这种方法不仅对Ca 2 +成像，但也可以适应任何实时成像，而每行为观察。例如，我们可以访问任何通过细胞使用电极直接记录活动。结合双光子激发显微镜，我们是能够执行的时间时隔突触结构成像，它可以与行为改变。因此，这种方法与行为观察大脑成像神经网络的功能解剖不仅是宝贵的，但可以作为一个强大的工具，相关的突触可塑性的14记忆背后的机制。

<table border="1"><tbody><tr><td> 设备/试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> tetric EvoFlow </td><td>义获嘉Vivadent </td><td> M04115 </td><td>光固化胶</td></tr><tr><td> BX51WI显微镜</td><td>奥林巴斯</td><td> BX51WI </td><td>随着40X（NA 0.8）浸水物镜</td></tr><tr><td>旋转盘共聚焦显微镜系统</td><td> Improvision在珀金埃尔默</td><td></td><td>与491 nm激光</td></tr><tr><td>体视显微镜</td><td>尼康</td><td> SMZ-800 </td><td>连接到摆臂。立体声的观点仍然是录像</td></tr><tr><td>雁皮，和纸</td><td>灰原，日本</td><td></td><td>一种特殊的日本传统纸</td></tr><tr><td> CCD相机</td><td>影像来源</td><td> DFK41AU02 </td><td> 1/2“1080×960像素的SONY CCD </td></tr><tr><td>操纵杆操纵</td><td> narishige </td><td> MN-151 </td><td></td></tr><tr><td>注射器</td><td> narishige </td><td> IM - 5B </td><td></td></tr></tbody></table>

simultaneous recording of calcium signals from identified neurons and feeding behavior of drosophila melanogaster

他们在行为的功能研究的神经网络，我们必须分析神经元是如何运作时产生的每一个行为模式。因此，神经活动和行为的同时录音是必不可少的行为相关的大脑活动。对于这样的行为分析，果蝇， 果蝇 ，让我们纳入如GCaMP 1基因编码的钙指标，监测神经元的活动，并使用为optogenetic或thermogenetic技术复杂的遗传操作，特别激活确定神经元2-5。使用一个thermogenetic技术已经使我们发现摄食行为的关键神经元（洪水等，正在修订）。作为摄食行为的主要组成部分， 果蝇成年喂养6（长鼻延伸反应; PER）的延伸，它的长鼻，从它的长鼻或跗骨的感觉细胞的甜刺激的响应。合作每7 mbining协议与钙成像技术，使用GCaMP3.0 1 8日，9时 ，我已经建立了一个实验系统，在这里我们可以监视在摄食中枢的神经元活动- suboesophageal节（SOG），同时与行为观察长鼻。我已经设计了一个仪器（“苍蝇的大脑实时成像和电舞台”：“苍蝇”），以适应一个果蝇成人，允许其长鼻自由移动，而它的大脑暴露洗澡钙通过浸水镜头+成像。苍蝇也适用于多种类型的现场苍蝇的大脑，如电生理记录或突触形态时移成像实验。由于实时成像结果可直接相关，同时每行为，这种方法可以提供一个很好的实验系统研究神经元网络的信息处理，以及如何这个Cellular活动耦合塑料进程和内存的。

Watch this Scientific Journal Video about Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster at JoVE.com

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

果蝇，<em&gt;果蝇</em&gt;，延长喂养它的长鼻，从长鼻或睑板糖刺激。我有长鼻延伸（PER）的响应与钙成像技术相结合的意见，使我们能够监测活动在大脑中的神经元，同时与行为观察。

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, ...

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

15.5K 次观看.  University of Massachusetts Medical School. 果蝇，果蝇，延长喂养它的长鼻，从长鼻或睑板糖刺激。我有长鼻延伸（PER）的响应与钙成像技术相结合的意见，使我们能够监测活动在大脑中的神经元，同时与行为观察。

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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

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 ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.