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 width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

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 ...

in-vitro-characterization-electrophysiological-properties-colonic

Research

JoVE Journal

Neuroscience

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

6.8K 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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

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 ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

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 ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由游泳鱼中可移动的特龙对神经元的无线电生理记录

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...