0920060 NSF资助，NIH资助DC004395 NHMRC赠款1009482，科学和医学研究的新南威尔士州办事处，加内特过时和罗德尼·威廉斯纪念基金会的支持。

1。头部约束概述<ol><li>组装一个定制的头后 ，插入1/16“不锈钢辊针直刺入钻了一个洞在3/32”不锈钢杆形成一个十字架。头后的垂直一块应该是约20毫米和跨一块约15毫米的水平。点击垂直杆的一端接受＃1-72螺钉。 （ 图1）。 </li><li>在手术过程中及录音，头后固定在一个定制的黄铜或铝安装酒吧 ＃1-72螺丝（ 图2）。安装杆的长片，应该是约90毫米长和宽5-10毫米。适合对开长条，角度为45度15毫米扩展成槽切头后。为了防止头部转动，头部后紧贴横梁上安装杆底部的小槽。 </li><li>越来越多的酒吧，然后连接到一个特制的铝安装集团K（ 图3）。安装块大约为30毫米×30毫米×25毫米。这种安装块附着到微操作机器人，使头后的位置，可以准确地沿中线放置于前囟门。两件一起安装用螺丝安装块。一个块中切出允许安装杆滑动，并用螺钉固定。安装酒吧台面平行。在录制过程中，争取在一个定制的立体框架（ 图4）酒吧。设计stereotax是安装在相同的位置在手术过程中在安装块吧。这样的安排，确保鼠标将始终对准跨越实验会话立体框架。 </li><li>钨棒（直径0.010“），以削减约5毫米的长度，约1毫米弯曲45°。这杆为接地脚（ 图1）。 </li></ol> 2。立体铝ignment为开颅手术 注意：下面列出的所有程序按照标准的无菌手术技术已经由华盛顿州立大学动物护理和使用委员会和加尔文研究所动物伦理委员会批准。 <ol><li>在感应室内放置5％异氟醚麻醉的小鼠。尾巴和/或脚趾掐反应缺乏确保该动物是完全麻醉。 </li><li>将鼠标鼠标适配器（Stoelting）的装备在啮齿类动物立体框架。鼠标的方向正确，将里面的牙齿咬栏孔，并拧紧，所以它是紧贴鼻夹。将鼠标作为直咬酒吧和鼻钳可能。将制成的乳胶手套，一块鼠标的鼻子，口罩，并保持在5％的异氟醚，直到鼠标的呼吸率约为1呼吸/秒。关闭异氟醚1.5 - 2.0％。固定头，采用t他耳酒吧小心不要刺破耳膜。酒吧应该是贴身但不渗入耳道太深。 </li><li>鼠标下方放置一个加热垫。 </li><li>申请眼药膏，以防止眼睛干燥。 </li><li>剃的头皮之间的耳朵小剃须刀或细点的剪刀眼睛。 </li><li>清洗和消毒氯碱hexidine（或优碘），然后用酒精擦洗冲洗3次重复擦拭头皮。 </li><li>来打开颅骨顶部的皮肤做一个切口，沿中线从后脑勺眼睛。 </li><li>用手术刀刮去骨膜切口边缘。 （根据实验目标）如果需要的话，仔细地收回镊子颈部背面的肌肉，使肌肉沿束撕裂，以减少出血。适用于头皮组织下面的明胶海绵，以减少水分，防止皮肤从头骨。 </li><li>干颅骨表面用棉签蘸异丙醇，以提高可视化颅骨地标。 </li><li>将头骨标准立体坐标13。罚款点探针插入电极支架和重视它的立体定向显微操作。一个尖锐的金属棒工作以及为此目的（ 图5）。 </li><li>使用点，以确定头骨上的囟门（ 图5A）和Lambda（ 图5B）。这些点之间来回移动探头，检查每个位置的内侧外侧不超过50微米的坐标不同。需要仔细松动耳酒吧，横向转移的头部和耳朵酒吧，并重新拧紧耳酒吧定位头部。 </li><li>确认在每个位置的背腹的高度不超过50微米的不同。调整鼻钳的高度，这将旋转轴耳酒吧周围的头骨，平的头骨，如果有必要的间距。耳杆的高度，可能还需要进行调整。 </li><li>使用地图集13日确定利益的目标（ 图5C）相对囟坐标。上述目标的头骨（例如，一个“X”标记，或用墨汁点四方形的角落）将开颅手术（ 图5D）。 </li></ol> 3。头后和接地引脚安装<ol><li>使用蚀刻凝胶清洁头骨。使用撒施擦洗和遍布表面蚀刻胶（10秒）。用清水洗净，完全晾干。适用底漆牙科水泥颅骨表面采用撒施。传播新喷头的粘合剂。固化胶UV光枪（1周期）。 </li><li>选择一个远端目标站点的接地引脚的位置。孔在地面p的短端使用一个＃65 miniblade手术刀的一角，也足够宽头骨仔细一小孔在进入和休息后，脑膜。接地引脚应为导向，以便它指向前囟门和未来的头后（ 图6A）。 </li><li>头后固定安装的酒吧和封锁和附加的微操作机器人。移动的微操作机器人在三个层面上，定位在头后，前囟门刚刚接触的头骨（ 图6B）。基地的头后应该趴在颅骨平。 </li><li>使用解剖探头，适用于头后和接地引脚（ 图6C），周围有少量的牙科水泥。按水泥，以确保良好的接触与头骨。注意：不要使用过多的水泥或不会变硬均匀。防治紫外光枪（3-4周期从多个角度）的水泥。 </li><li>适用于水泥的第二阶段和周围的头部后建立的基础和覆盖的头后的沟槽（ 图6D）。 </li></ol> 4。开颅手术<ol><li>找到了开颅手术的参考标志，这将部分底漆掩盖。使用65＃手术刀，以纪念3毫米×3平方毫米围绕这个站点和应用利多卡因下降，降低灵敏度，以减少出血。 </li><li>使每个广场的一侧（多达10个）多得分，小心不要通过骨骼和削减到大脑（ 图7A）。 </li><li>一旦骨瓣松动，使用手术刀的一角撬起来，离开硬脑膜完好（ 图7B）。 </li><li>用骨蜡覆盖。 </li></ol> 5。手术后<ol><li>关闭气体麻醉和从鼠标的立体框架。 </li><li>用棉签申请周围裸露的皮肤利多卡因软膏，以缓和伤口周围的轰动。使用另一种棉签应用抗生素软膏裸露的皮肤。注入酮洛芬（5毫克/公斤的IM或IP基于个人IACUC兽医偏好的）手术后镇痛。鼠标一般会和天然气停止分钟内移动。 </li><li>将鼠标在一个温暖的笼子和显示器直到它准备返回到动物的住房。 </li><li>动物应每天检查至少一次后，它已经从手术中恢复。观看不良的饮食和/或饮酒和无精打采行为的迹象。如果怀疑疼痛，直到缓解酮洛芬每24小时（在手术后相同剂量）。利多卡因局部可应用的伤口，如果动物被刮伤或显示不适的迹象。动物一般恢复手术，无并发症或疼痛。 </li></ol> 6。录音和染料注射液<ol><li>等待后，头部手术后至少一天前使用鼠标录音。 </li><li>塑造地方在泡沫束缚设备的鼠标鼠标的身体。鼠标已经被改编到该设备，从而减少其焦虑。典型的适应断奶后大约一个星期前，手术和实验小鼠每天处理，放置在发泡设备鼠标，时间为1-5分钟。暂停在立体框架（ 图4）的设备。 </li><li>安全的头后安装吧，这是现在固定的立体框架（ 图4）。 </li><li>用生硬的22号注射器针头尖端终止线连接到接地引脚。 </li><li>在开颅取出骨蜡。取出硬脑膜如果有必要使用一个miniblade手术刀尖端。 </li><li>驱动电极，以适当的立体位置，并开始录制（ 图8）。只有电生理记录，各种电极都可以使用。电极的选择是需要录音的位置和类型的依​​赖。清醒鼠标录音电极的选择将是什么研究员在麻醉鼠标使用相同。对于合并的电生理记录染料/示踪剂注射，微量电极使用10。微量充满染料/示踪剂的选择，以便它可以被微电泳注入的电生理实验结束。电极也可用于多管，这样可以药理操纵。多管电极安装在一个单一的录音微量（ 图8）。这些技术标准文献和麻醉与清醒的录音是不是不同。 </li><li>录音可为4-5个小时，连续超过3-4天。 </li><li>在录制会议结束时，染料/示踪剂可以注射。因为有没有在大脑疼痛受体，离子导入或进入大脑的染料示踪注射是不可能造成的不适。然而，一次注射是该地区的电反应特性的特点，动物，可能是麻醉异氟醚f或注射。这将消除任何潜在的不必要的感觉。因为染料/示踪已经在微管，可以主动和地线接通的电生理记录设置的电流发生器，微电泳注入染料/示踪。使用适当的协议，在电极的染料/示踪。删除从headpost克制和身体克制的动物，并返回到其家乡笼。 </li><li>在实验结束后，按照适当的协议安乐死和组织处理，以恢复注射部位和标签。 </li></ol> 7。代表结果成功安装的头后，允许实验者记录单，多单元的响应，从麻醉的，清醒的鼠标多天。约束系统可以稳定在大脑中单个神经元的电生理记录。极好的隔离单的单位和强烈反响ţØ刺激，可记录多天从相同的大脑结构，例如在小鼠下丘（ 图9A，日图9B，一天两）。带了一个好头安装后，将始终对准每个鼠标的立体定位仪和鼠标立体定向图谱，13，特别是脑细胞的可靠定位。这个可靠的定位，使染料和示踪剂注入后多个录音天的具体结构。例如，评估降预测从听觉中脑听觉脑干，生物素葡聚糖胺（可视作为chromagen使用的二氨基联苯胺）后，在注射部位的神经元的反应特性电生理识别，可以注射到小鼠下丘（ 图10A） （ 图10A，一个神经元与最佳频率为51千赫），组织处理后，对侧耳蜗核背侧nterograde标签可以被可视化和策划（ 图10B）。显微照片也可以得到更高的分辨率，查看顺标轴突和终端（ 图10C）。 <img alt="图1" src="/files/ftp_upload/3755/3755fig1.jpg" /> 图1。 <img alt="图2" src="/files/ftp_upload/3755/3755fig2.jpg" /> 图2。 <img alt="图3" src="/files/ftp_upload/3755/3755fig3.jpg" /> 图3。 <img alt="图4" src="/files/ftp_upload/3755/3755fig4.jpg" /> 图4。 <img alt="图5" src="/files/ftp_upload/3755/3755fig5.jpg" /> 图5。 <img alt="图6" src="/files/ftp_upload/3755/3755fig6.jpg" /> <s阮富仲&gt;图6。 <img alt="图7" src="/files/ftp_upload/3755/3755fig7.jpg" /> 图7。 <img alt="图8" src="/files/ftp_upload/3755/3755fig8.jpg" /> 图8。 <img alt="图9" src="/files/ftp_upload/3755/3755fig9.jpg" /> 图9。 <img alt="图10" src="/files/ftp_upload/3755/3755fig10.jpg" /> 图10。

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我们什么都没有透露。

后头部约束系统的电生理及神经解剖实验小鼠的优势是可以进行麻醉的，清醒小鼠实验，消除潜在的响应由于麻醉药品的污染。此外，一套最多可以使用多天，以便更有效地使用动物。 头后的大部分组件是可用的现成的。只有头部后和安装设备的定制，这两者是相对简单的建设，是可以重复使用。如果一台机器的店是不是在一个机构，研究者可能使用<a href="http://www.emachineshop...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>牙科水泥前清洗头骨的蚀刻凝胶 </td><td>亨利施恩</td><td> 101-5396 </td><td> 12/pk用1.2毫升注射器</td></tr><tr><td>牙科水泥底漆 </td><td>克尔Optibond </td><td> 25881 </td><td> 8毫升瓶装</td></tr><tr><td>牙科水泥胶粘剂 </td><td>克尔Optibond </td><td> 25882 </td><td> 8毫升瓶装</td></tr><tr><td> 喷头的牙科水泥</td><td>克尔Optibond </td><td> 24680 </td><td> 200/pk </td></tr><tr><td> 牙科水泥 </td><td>魅力，贺利氏古莎</td><td> 66000085 </td><td> 4gm注射器笔芯</td></tr><tr><td>钨棒（ 接地引脚 ） </td><td>上午系统</TD&gt; <td> 717200 </td><td> 0.010“直径</td></tr><tr><td>经济紫外线光固化 </td><td>亨利施恩</td><td> CU-80 </td><td></td></tr><tr><td> 头后 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 安装酒吧 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 固定块 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 立体框架 </td><td>大卫Kopf仪器</td><td> 902 </td><td></td></tr><tr><td> 鼠标和新生大鼠适配器 </td><td> Stoelting </td><td> 51625 </td><td></td></tr><tr><td> Deltaphase恒温垫 </td><td>布伦特里科学公司</td><td> 39DP </td><td></td></tr></tbody></table>

preparation of an awake mouse for recording neural responses and injecting tracers

这是众所周知的麻醉，神经反应特性改变在大脑的不同区域。13。在听觉系统，脑干神经元，包括阈值，频率特异性，抑制边带根本性的响应特性，在麻醉下1-2显着的方式被改变。这些观察提示生理学家设法从单个神经元的记录，没有麻醉的污染影响。其结果之一是去大脑的准备，完全是在中脑4水平横断脑干。该制剂的弊端，是一项艰巨的手术，消除降的预测从脑，无法用感官刺激，检查以上的脑结构。不同的策略已植入电极阵列，长期记录单个神经元和多单元集群，而动物是清醒和/或行为5,6 </suP&gt;。然而，这些技术与示踪染料注射后，电生理特征的大脑结构不兼容。为了避免改变与麻醉药的神经反应性能，同时记录单个神经元的电生理反应特性，我们已经适应了长期用鼠标10-12蝙蝠7-9头部约束技术。使用这种方法，我们能够在麻醉的小鼠进行了几天的电生理记录。我们可以在录音结束，然后注入一种染料，重建电极位置和记录点，或注入示踪剂，所以可确定，并从记录位点的途径。这种方法无需使用麻醉剂以及隔离多天的单神经元记录。

Watch this Scientific Journal Video about Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers at JoVE.com

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

重要的是理解大脑功能和指导安置途径示踪染料神经反应的电特性。然而，许多研究都在麻醉动物。理解没有麻醉药的脑功能，我们开发了一种方法来记录神经元的反应特性，并在清醒小鼠注入染料。

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

preparation-an-awake-mouse-for-recording-neural-responses-injecting

Research

JoVE Journal

Neuroscience

19.3K 次观看.  Johns Hopkins University. 重要的是理解大脑功能和指导安置途径示踪染料神经反应的电特性。然而，许多研究都在麻醉动物。理解没有麻醉药的脑功能，我们开发了一种方法来记录神经元的反应特性，并在清醒小鼠注入染料。

视频: Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

科研

神经科学

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

高接触密度的制作，平面接口神经电极用于记录和刺激的应用

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

一种改进的多电子四极超光速引擎在行为大鼠大尺度神经记录中的构建

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...