Name: in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats
Uploaded: 2017-09-27T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

该议定书得到了中国国家自然科学基金 (#31171066、#81270464) 和中德科学中心 (GZ919) 的研究资助。

这里报告的实验协议已获得上海交通大学动物伦理委员会 (SYXK2013-0050) 的批准. #160; 完整的神经节和神经直肠的解剖躯干需要至少15分钟的人在这项技术相当经验。因此, 有必要保持动物活着, 但在深麻醉下, 同时执行解剖, 以确保组织的可行性, 随后的电生理记录. 
 1. 灌注液和试验化合物的制备 <ol> <li> 准备 5 L 的克雷布斯溶液: 113 mm 氯化钠, 5.9 毫米氯化钾, 1.2 mm NaH 2 PO 4 , 1.2 mm MgSO 4 , 25 mm NaHCO 3 , 1.2 mm CaCl 2, 11.5 毫米葡萄糖。使用 95% O 2 + 5% CO 2 气体混合物饱和解决方案。预冷〜500毫升的含氧克雷布斯在冰箱. </li>
	<li> 在需要时, 准备等分的原料测试化合物 (1 毫米的乙醇和 10 mm 5-色 (5-HT) 中的辣椒素)。稀释的股票在克雷布斯 (用于浴应用) 和盐水 (为腔内管理) 到最后的浓度, 才使用前. </li>
</ol> 2。记录电极的制备 <ol> <li> 使用常规电极拉拔器从标准玻璃管中拉出无内丝 (1.5 mm 外径) 的记录电极。调整拉拔器设置的热量和拉力, 使被拉扯的电极的小腿在20和25毫米之间. </li>
</ol> 3。组织集合 <ol> <li> 使用戊巴比妥钠 (80 毫克/千克, ip) 深麻醉大鼠。和 #160; </li> <li> 在确保不育的同时, 通过使用手术刀在腹壁上进行中线切口来暴露腹腔。把肠系膜和其他组织拉到一旁, 露出直肠. </li>
	<li> 将动物置于解剖显微镜下。通过仔细解剖, 找到左 PG, 并确定 PN 和 LSN 加入它。把这些神经从 PG 上剪掉几毫米. 
	注: PG 位于结直肠交界处附近。通常, 3-4 PN 从腰骶脊髓和一个 LSN 项目到 PG ( 图 1 ). </li>
	<li> 切断耻骨骨以暴露直肠。移除直肠以上的组织 ( 即, 膀胱, 等 );小心把 PG 保持原样. </li>
	<li> 通过静脉注射戊巴比妥过量而牺牲老鼠。在 PG 上方约3厘米的结肠横断面上, 用镊子将直肠从动物身上取出. </li>
	<li> 将直肠转移到填充预克雷布斯溶液的培养皿中。通过轻轻冲洗结肠来去除粪便。小心去除残留的膀胱和其他组织, 而不损害 PG. </li>
</ol> 4。结肠传入神经的解剖 <ol> <li> 将冒号放入记录室 (20 毫升), 并连续灌注 carbogenated 克雷布斯溶液。设置15毫升/分钟的灌注率 </li>
	<li> Cannulate 直肠在口腔和肛门两端。开始结肠腔内灌注生理盐水的速度为10毫升/小时在口腔肛门的方向. </li>
	<li> 在解剖显微镜下定位主要 PG。使用昆虫引脚暴露神经节。仔细解剖后, 找到神经节发出的细枝, 向结肠跑去 ( 图 1 )。切断神经节的距离. 
	注意: 图 1 说明了从远端的神经分支到 PG 的记录。神经可能包含骨盆和腰椎内脏传入纤维的混合物。或者, 可以从 PN 和/或在 PG 的 LSN 上进行记录. </li>
	<li> 打开加热浴缸并预热克雷布斯溶液以保持室温在34和 #177; 0.5 和 #176; C. </li>
</ol> 5。吸入电极的制备 <ol> <li> 采取 pre-pulled 玻璃吸管 (步骤 2) 并在解剖显微镜下检查。用一对镊子打破电极的尖端, 使其尺寸与要记录的神经直径相兼容. </li>
	<li> 将笔尖放在较亮的光晕附近, 以将其倾斜. </li>
</ol> 6。电生理记录 <ol> <li> 将斜面电极连接至电极支架。将10毫升注射器连接到托架上的侧面端口, 以对电极施加负或正向压力. </li>
	<li> 将刀柄连接至 bioamplifier 的 headstage, 并将 headstage 安装到机械手上. </li>
	<li> 将电极移到组织浴中, 用温和的吸力将电极克雷布斯溶液, 直到接触到支架的银线。将电极尖端靠近神经的切口端, 施加负压以将神经吸入电极。施加更多的负压, 使 ~ 1 毫米的神经被拉入电极, 形成一个紧密的密封. </li>
	<li> 打开 bioamplifier, 并将滤镜设置为 300-3000 Hz. 监视示波器上的信号, 并使用带有峰值数据处理的计算机记录神经信号 (20 kHz 采样率) 和腔内压力信号 (100 赫兹采样率)软件. </li>
</ol> 7。测试结肠传入敏感度 <ol> <li> 通过关闭插座套管上的三路水龙头, 同时连续注入 intraluminally, 从而应用结肠的斜坡扩张。监测腔内压力, 直到它达到60柱, 在这个时候打开三路水龙头的引流套管. </li>
	<li> 定期重复此过程15分钟. 应用药物 extra-or intraluminally 测试传入神经的化学敏感性. </li>
</ol>

<ol>
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作者声明没有利益冲突。

该协议是一个相对简单的实验方法来评估大鼠结肠传入的电生理特性。该协议 (从组织剥离到建立神经记录) 通常需要大约2小时才能完成。组织收集 (步骤 3) 和吸入电极 (步骤 5) 的制备是关键步骤。这是至关重要的, 能够找到 PG, LSN 和 PN, 并注意不要破坏神经节和神经组织解剖。玻璃吸管的尖端必须被打破和斜面到一个大小兼容的神经束。
该制剂通常是可行的几个小时, 允许研...

胃肠道 (GIT) 是丰富的支配与外部传入神经, 传达感官信号从肠道到中枢神经系统, 这有助于肠道-大脑的相互作用。改变兴奋性的这些外在传入, 以及改变中央处理的传入输入, 基础内脏疼痛和其他症状的胃肠条件, 包括功能性和炎症性肠道疾病1。来自直肠的感官信息主要通过胸腰椎/下和腰骶/盆腔神经 (PN)2传达。在啮齿动物疾病模型中, 对这些初级传入纤维的电生理特性的研究有了更大的兴趣。然而,在体内啮齿类动物结肠传入的电生理记录是一个技术挑战, 需要相当多的手术技巧。此外, 血液动力学的变化, 组织运动和麻醉药也可能影响神经活动和敏感性测试刺激在体内。因此, 近年来, 越来越多的研究采用了体外(前体内) 不同物种 (包括小鼠、大鼠、豚鼠和人类) 的制剂, 来研究结肠的感觉传导机制。传入和改变的兴奋性在疾病情况下。3,4,5,6,7,8
两种类型的前体结肠准备主要报告了: "平板" 准备5,9,10和 "管" 准备3,4。"平板" 小鼠直肠准备的视频协议以前已发布11。在本协议中, 小鼠直肠, 与 PN) 或腰椎内脏神经 (LSN) 附着, 是收获和 superfused 在一个组织室。直肠纵向切开, 将神经束延伸到充满石蜡油的记录室。使用单铂铱电极记录神经活动。该协议允许通过使用无偏电刺激来识别单个传入纤维的接受场。它本地化应用化学刺激, 以及应用不同的机械刺激范式 (例如,局灶性黏膜探查和周向伸展), 以传入神经末梢。因为神经必须从组织室延伸到一个单独的室, 所以保持附着的神经相对长是至关重要的;成功的神经解剖对这种方法的新的人构成挑战。最近, Nullens et al.发布了一个视频协议, 用于在小鼠空肠和结肠段 12中进行肠系膜传入的记录。在这个 "管" 的准备, 肠段与肠系膜附着保持完好, 从而允许分级扩张和 extra-luminal 管理不同的化学品。由于肠系膜神经被记录使用吸入电极, 它可以定位于组织附近, 即使肠系膜神经相对较短, 也可以记录传入活动。然而, 肠系膜神经包括迷走神经和脊髓传入纤维的混合种群, 支配空肠或胸腰椎下。腰骶部盆腔传入支配直肠, 不能在本协议中受到歧视。在这里, 我们提出了一个详细的协议, 对大鼠结肠传入的电生理记录使用 "管" 直肠制剂与完整的 PG。此方法可用于描述腰椎内脏 (下) 和腰骶盆腔传入的功能特性。

<table><tbody><tr><td>Sodium Pentobarbital</td><td>Shanghai Westang Bio-Tech</td><td>B558</td><td></td></tr><tr><td>Capsaicin</td><td>Sigma</td><td>M2028</td><td></td></tr><tr><td>Electrode puller</td><td>MicroData Instrument Inc</td><td>PMP107</td><td></td></tr><tr><td>Neurolog System (Bioamplifier)</td><td>Digitimer, Ltd</td><td>Neurolog System</td><td></td></tr><tr><td>A/D converter</td><td>Cambridge Electronic Design</td><td>Micro1401</td><td></td></tr><tr><td>Data processing software</td><td>Cambridge Electronic Design</td><td>Spike2 version 6</td><td></td></tr><tr><td>Silver wire</td><td>World Precision Instruments</td><td>EP12</td><td></td></tr><tr><td>Glass tubes</td><td>World Precision Instruments</td><td>1B150-4</td><td></td></tr><tr><td>Electrode holder</td><td>World Precision Instruments</td><td>MEH3SBW</td><td></td></tr><tr><td>Heating bath</td><td>Grant</td><td>GR150</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>Zoom2000</td><td></td></tr><tr><td>Dissecting microscope</td><td>World Precision Instruments</td><td>PZMIII-BS</td><td></td></tr><tr><td>Cigarette lighter</td><td>any</td><td>NA</td><td></td></tr><tr><td>Surgical tools</td><td>World Precision Instruments</td><td>NA</td><td></td></tr><tr><td>Insect pins</td><td>home-made from 0.1 mm stainless steel wire</td><td>NA</td><td></td></tr><tr><td>Three way manipulator</td><td>World Precision Instruments</td><td>KITF-R</td><td></td></tr><tr><td>Rats</td><td>Any</td><td>NA</td><td>Any strain/sex can be used.</td></tr></tbody></table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

结肠感觉神经功能障碍已被牵连的病理生理学的几个常见的条件, 包括功能性和炎症性肠道疾病和糖尿病。在这里, 我们描述了一个协议的体外结肠传入在大鼠的电生理特性的表征。直肠, 与完整的盆腔神经节 (PG) 连接, 从鼠身上移除;superfused 与 carbogenated 克雷布斯溶液在记录室中;和中空的口腔和肛门两端允许扩张。从 PG 中产生的细神经束被识别, 并使用吸入电极记录多传入神经活动。结肠段的扩张引起多放电的逐渐增加。对低、high-threshold 和宽动态范围传入光纤进行了主成分分析。结肠传入的化学敏感性可通过对试验化合物的浴或腔内管理进行研究。本议定书可以修改, 以适用于其他物种, 如小鼠和豚鼠, 并研究不同的电生理特性的胸腰椎/下和腰骶/盆腔传入的下降结肠正常和病理条件。

图 1是为ex 体内"管" 直肠准备的实验性设置的示意图, 其中有代表从远端神经到 PG 的记录。神经可能包含骨盆和腰椎内脏传入的混合物。在正常大鼠的准备中, 结肠传入神经通常有不规则自发活动的低水平。结肠的斜坡扩张导致射击速度的逐渐增加 (图 1B)。通过绘制腔内压力-传入神经反应曲线 (图 1C...

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In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

异常的感官功能是内脏疼痛和其他功能性和炎症性肠道疾病的症状。本文介绍了一种在前体大鼠直肠制备中结肠传入神经的电生理记录的协议。

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

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Neuroscience

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

6.9K 次观看.  Shanghai Jiaotong University School of Medicine. 异常的感官功能是内脏疼痛和其他功能性和炎症性肠道疾病的症状。本文介绍了一种在前体大鼠直肠制备中结肠传入神经的电生理记录的协议。

视频: In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

科研

神经科学

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

医学

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

在鼠标空肠和结肠段肠系膜传入神经活性的体外记录

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://app.jove.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

离体大鼠结肠中结肠传入神经的电生理记录

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

Neurophysiology

Electrophysiology Techniques

In Vivo Single-Fiber Recording of Sciatic Nerve C-Fibers in Rats

Source: Mao, H., et al. <a href="https://app.jove.com/v/59234">Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure.</a> J. Vis. Exp. (2019).
This video demonstrates a single-fiber recording of C-fiber nerve conduction in rats. An anesthetized rat is incised to expose the sciatic nerve, which provides sensory and motor supply to the lower limbs. The individual C-fibers are then exposed. Electrical stimulation is provided using stimulus electrodes implanted in the foot, and a response is recorded from a single nerve fiber.

大鼠坐骨神经 C 纤维的体内单纤维记录

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.