Name: in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats
Uploaded: 2017-09-27T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

이 프로토콜은 국가 자연 과학 재단의 중국 (#31171066, #81270464)와 중국-독일어 과학 센터 (GZ919)에서 연구 보조금에 의해 지원 되었다.

여기에서 보고 된 실험 프로토콜은 동물 윤리 위원회의 상하이 교통 대학의과 대학 (# SYXK2013-0050)에 의해 승인 되었습니다. 그대로 신경 절 및 신경 colorectum의 해 부 트렁크는이 기술에 아주 경험 있는 사람에 대 한 최소 15 분 걸립니다. 그것은 깊은 마 취 동물 살아 있지만 해 electrophysiological 기록에 대 한 조직의 생존을 보장 하기 위해 수행 하는 동안 유지 하는 데 필요한 따라서.
 1. 관류 솔루션의 준비 및 시험 화합물 <ol><li>준비 5 L 크레브스 솔루션의: 113 mM NaCl, 5.9 m m KCl, 1.2 m m NaH 2 포 4, 1.2 m m MgSO 4, 25 m m NaHCO 3, 1.2 m m CaCl 2 , 및 11.5 m m 포도 당. 95% O 2 + 5% CO 2 가스 혼합 솔루션을 포화. 미리 냉장고에 산소 Krebs의 ~ 500 mL를 냉각.</li>
	<li>준비 aliquots 재고의 필요에 따라 화합물 (에탄올과 10 m m 5-hydroxytryptamine (5-HT) 염 분에에서 1 mM capsaicin)를 테스트 합니다. 그냥 사용 하기 전에 최종 농도에 Krebs (목욕 용)와 염 분 (intraluminal 관리)에 대 한 재고 희석.</li>
</ol> 2. 기록 전극의 준비 <ol><li>표준 유리에서 기록 전극 없이 기존의 전극 끌어당기는 사람을 사용 하 여 내부 필 (1.5 m m 외부 직경) 튜브 풀. 열에 대 한 끌어당기는 설정을 조정 하 고 당기는 가져온된 전극의 칼 20 사이 25 m m.</li>
</ol> 3. 조직 컬렉션 <ol><li>Anaesthetize 나트륨 pentobarbital (80 mg/kg, i.p.)를 사용 하 여 깊이 쥐. </li> <li>무 균을 보장 하면서 메스를 사용 하 여 복 부 벽에 midline 절 개를 수행 하 여 복 부 구멍을 노출. mesentery 및 옆으로 colorectum를 폭로 다른 조직.</li>
	<li>해 현미경 동물을 배치합니다. 조심 해 부를 통해 왼쪽된 PG 찾아서는 PN과 합류 하는 LSN을 식별 합니다. 이 신경은 페이지 멀리 몇 밀리미터 잘라 
	참고: PG 장 교차점에 가까운 속 인 다. 일반적으로 3-4 PN lumbosacral 척수 및 LSN 프로젝트에서 PG ( 그림 1)를.</li>
	<li>항문 노출 관절 뼈를 잘라. Colorectum; 위의 조직 (즉, 오 줌 방광, 등) 제거 PG를 그대로 조심.</li>
	<li>는 Pentobarbital의 과다 복용의 정 맥 주입 하 여 쥐를 희생. 콜론 세 위에 약 3cm transect 고 집게를 사용 하 여 동물에서 제거 하는 colorectum.</li>
	<li>사전 냉각된 Krebs 솔루션으로 가득 페 트리 접시에는 colorectum를 전송 합니다. 부드럽게 콜론을 플러시하는 배설물을 제거 합니다. 남은 오 줌 방광 및 다른 조직에 페이지를 손상 없이 신중 하 게, 제거</li>
</ol> 4. 저 승 구심 성 신경의 해 부 <ol><li>콜론 녹음 챔버 (20 mL)로 놓고 perfuse carbogenated Krebs 솔루션을 지속적으로 조직. 15 mL/분에서 관류 속도 설정</li>
	<li>구강과 항문 끝에 colorectum cannulate. 염 분에서 항문 방향으로 구두 10 mL/h의 속도로 콜론의 intraluminal 주입 시작.</li>
	<li>해 현미경 주요 PG를 찾습니다. 곤충 핀을 사용 하 여 노출 된 신경 절. 조심 해 부, 신경은 신경 절에서 유출 하 고 콜론 ( 그림 1)으로 실행의 좋은 지점을 찾을. 가까이 신경 절의 신경 절단. 
	참고: 그림 1에 나와 있는 페이지에 말 초 신경 지점에서 녹음 신경은 골반과 요 추 splanchnic 구심 성 섬유의 혼합물을 아마도 포함 되어 있습니다. 또는, 녹음은 PN 및 LSN 인접 하는 페이지에서 만들어질 수 있다</li>
	<li>난방 목욕과 따뜻한 34 ± 0.5 ° c.에 챔버 온도 유지에 Krebs 솔루션 설정</li>
</ol> 5. 흡입 전극의 준비 <ol><li>미리 가져온된 유리 피 펫 (2 단계) 하 고 해 현미경 검사. 그것은 호환 기록 신경의 직경 크기의 집게의 쌍 전극의 팁을 부.</li>
	<li>라이터 플레어 가까이 배치 하 여 팁을 경사.</li>
</ol> 6. Electrophysiological 녹음 <ol><li>연결 전극 홀더를 경사진된 전극. 측면 포트 전극에 부정적 또는 긍정적인 압력을 적용 하려면 소유자에 10 mL 주사기를 연결.</li>
	<li>소유자는 bioamplifier의 headstage에 연결 하 고 조작자에 headstage를 탑재.</li>
	<li>조직 목욕 전극 이동한 소유자의 실버 와이어 연결 될 때까지 부드러운 흡입을 적용 하 여 전극 Krebs 솔루션을 작성 합니다. 신경의 절단된 끝 가까이 전극 팁 놓고 신경 전극으로 빨 아 부정적인 압력을 적용 합니다. ~ 1 m m는 신경의 전극으로 당겨 하 고 단단한 물개를 형성 더 부정적인 압력을 적용.</li>
	<li>회전 설정 필터 300 3000 Hz. 하 고 bioamplifier에 오실로스코프에 신호를 모니터링 하 고 기록 신경 신호 (20 kHz 샘플링 속도) 및 스파이크 데이터 처리와 컴퓨터를 사용 하 여 intraluminal 압력 신호 (100 Hz 샘플링 속도) 소프트웨어.</li>
</ol> 7. 저 승 성의 감도 테스트 지속적으로 intraluminally를 일으키는 동안 콘센트 정에 3 방향 탭을 닫아서 콜론의 <ol><li>적용 램프 팽창. 그것에 도달할 때까지 60 mmHg, 오픈 시간에 배출 정에 3 방향 탭 intraluminal 압력 모니터링.</li>
	<li>15 분 적용 약물 추가-또는 intraluminally의 구심 성 신경 화학 감도 테스트를 정기적으로이 절차를 반복.</li>
</ol>

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저자는 충돌의 관심을 선언합니다.

여기에 제시 된 프로토콜 쥐의 저 승 afferents의 electrophysiological 속성을 평가 하기 위해 비교적 간단한 실험 방법입니다. (신경 녹음을 설정 하 고 조직 해 부)에서 프로토콜 일반적으로 완료 하는 데 약 2 시간 걸립니다. 조직 컬렉션 (3 단계) 및 흡입 전극 (5 단계)의 준비는 중요 한 단계가 있습니다. 그것은 PG, LSN와 PN 주의 하지 신경 절 및 신경 조직의 해 부 동안에 손상 수 있을 중요 합니다. 유리 피...

위장 지역 (GIT) 풍부한 외부 구심 성 신경 중앙 신 경계를 용기에서 감각 신호를 전달 하 고 창 자-두뇌 상호 작용에 기여 innervated입니다. 이러한 외부 afferents의 변경 된 흥분 뿐만 아니라 구심 성 입력의 변경 된 중앙 처리 기본 내장 고통 및 다른 증상 기를 포함 한 조건 기능과 염증 성 장 질환1. 감각 정보는 colorectum에서 주로 thoracolumbar/하복과 lumbosacral/골반 신경 (PN)2를 통해 전달 됩니다. 공부 하는 설치류 질병 모델에서 이러한 기본 구심 성 섬유의 electrophysiological 속성에 대 한 관심 증가 되었습니다. 그러나, 설치류에 저 승 afferents의 비보에 electrophysiological 녹음 기술 도전 이며 상당한 수술 기술이 필요 합니다. 또한, hemodynamic 변화, 조직 운동, 그리고 마 취약 떨어질 수 있습니다 또한 신경 활동과 감도 비보에자극 테스트를. 따라서, 최근 몇 년 동안, 연구의 증가 고용 체 외에서 (ex vivo) 준비의 다른 종, 포함 쥐, 쥐, 기니 피그, 및 인간, 저 승에서 감각 전달의 메커니즘을 검토 하 afferents와 질병 상태에서 변경 된 흥분입니다. 3 , 4 , 5 , 6 , 7 , 8
두 가지 유형의 저 승 준비 비보 전 주로 보고 되었습니다: "플랫 시트" 준비5,,910 고 "튜브" 준비3,4. "플랫 시트" murine colorectum 준비를 위한 비디오 프로토콜11이전에 게시 되었습니다. 이 프로토콜은 PN과 마우스 colorectum), 요 추 splanchnic 신경 (LSN) 조직 챔버에 수확 하 고 superfused 또는. colorectum 경도, 오픈 절단 그리고 신경 번들 파라핀 기름을 가득 녹음 구획으로 확장 됩니다. 신경 활동 monopolar 백 금-이리듐 전극을 사용 하 여 기록 됩니다. 프로토콜 편견된 전기 자극을 사용 하 여 개별 구심 성 섬유의 수용 필드의 식별에 대 한 수 있습니다. 그것은 localizes 화학 자극의 응용 프로그램 뿐만 아니라 다른 기계적 자극 패러다임의 응용 프로그램 (예를 들어, 초점 점 막 프로 빙 및 원주 스트레치), 구심 신경 엔딩. 그것은 비교적 긴; 연결 된 신경 유지 하는 중요 한 신경 조직 약 실에서 별도 챔버를 확장 해야 합니다, 때문에 신경의 성공적인 절 개 그 도전이이 방법론에 새로운 포즈. 더 최근에, Nullens 그 외 여러분 murine jejunal에 저 승 세그먼트12mesenteric afferents의 생체 외에서 녹화를 위한 비디오 프로토콜을 출판. 이 "튜브" 준비에 연결 된 mesentery와 창 자 세그먼트 등급된 팽창 및 다른 화학 제품의 내부 및 추가 luminal 관리 되므로 그대로 유지 됩니다. Mesentery 신경 조직 가까이 배치 될 수 있습니다, 흡입 전극을 사용 하 여 기록 됩니다 이후 mesentery 신경은 상대적으로 짧은 하더라도 성의 활동을 기록할 수 있습니다. 그러나, mesentery 신경 자극 공장 vagal 및 척수 구심 섬유의 혼합된 인구 구성 또는 thoracolumbar 하복. Lumbosacral 골반 afferents 자극이 프로토콜에 차별 수 없습니다 colorectum. 여기, 선물이 electrophysiological 쥐 저 승 afferents "튜브" colorectum 준비는 그대로 페이지와 함께 사용 하 여 녹음에 대 한 자세한 프로토콜 이 방법은 splanchnic 요 추의 기능 특성의 특성에 대 한 수 있습니다 (하복) 및 lumbosacral 골반 afferents.

<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

저 승 감각 신경의 기능 장애 이상 기능 및 염증 성 대 장 질환과 당뇨병을 포함 하 여 몇 가지 일반적인 조건에 연루 되었습니다. 여기, 우리가 쥐에 있는 저 승 afferents의 electrophysiological 속성 생체 외에서 특성에 대 한 프로토콜을 설명합니다. 그대로 골반 신경 절 (PG),와 함께 colorectum; 쥐에서 제거 carbogenated 녹음 실;에서 Krebs 솔루션으로 superfused 그리고 팽창 수 있도록 구강과 항문 끝 cannulated. PG에서 나오는 미세 신경 번들을 식별 하 고 multiunit 구심 성 신경 활동 흡입 전극을 사용 하 여 기록 됩니다. 저 승 세그먼트의 팽창 multiunit에 점진적 증가 elicits. 주성분 분석은 낮은 임계값, 높은 임계값 및 넓은 동적 범위 구심 섬유 차별화 위해 수행 됩니다. 저 승 afferents의 화학 감도 시험 화합물의 목욕 또는 intraluminal 관리를 통해 공부 될 수 있다. 이 프로토콜은 다른 종, 쥐, 기니 피그, 등과 정상에서 하강 콜론의 thoracolumbar/하복 및 lumbosacral/골반 afferents의 electrophysiological 속성의 차이 연구 하는 응용 프로그램에 대 한 수정할 수 및 병 적인 조건입니다.

그림 1 은 비보 전 "튜브" colorectum 준비는 페이지에 말 초 신경에서 기록 하는 대표에 대 한 실험적인 체제의 구조 그림 신경은 아마도 골반과 요 추 splanchnic afferents의 혼합물을 포함. 정상 쥐에서 준비, 저 승 구심 성 신경은 일반적으로 불규칙 한 자발적인 활동의 저수준이 있다. 콜론의 램프 팽창 발사 속도 (그림 1B)에 있...

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

비정상적인 감각 기능 내장 고통 및 다른 증상 기능 및 염증 성 대 장 질환의 기초. 프로토콜에는 비보 전 쥐 colorectum 저 승 구심 성 신경의 electrophysiological 녹음에 대 한 여기에 제공 됩니다.

생체 외에서 쥐에 있는 저 승 구심 성 섬유의 Electrophysiological 속성의 특성

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

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JoVE Journal

Neuroscience

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

6.9K 조회수.  Shanghai Jiaotong University School of Medicine. 비정상적인 감각 기능 내장 고통 및 다른 증상 기능 및 염증 성 대 장 질환의 기초. 프로토콜에는 비보 전 쥐 colorectum 저 승 구심 성 신경의 electrophysiological 녹음에 대 한 여기에 제공 됩니다.

비디오: 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.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

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 ...

연구

신경과학

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.

손상 망막의 특성 Electrophysiological GFP - 표현 세포 집단

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.

마우스 대장 수입 성의 엔딩의 생체 기능 특성에

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

의학

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.

마우스 Jejunal과 대장 세그먼트에서 장간막 구 심성 신경 활동의 생체 외 녹음에서

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.

Vivo에서 Axonal 흥분 테스트 쥐 척 골 신경의 electrophysiological 측정

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.

전도 실패의 메커니즘을 검사하기 위해 연결된 Sciatic 신경을 가진 생체 내 단일 섬유 기록 및 손상되지 않은 등철 루트 신경절의 사용

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.

생체 외 쥐 결장에서 결장 구심성 신경의 전기생리학적 기록

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.

쥐에서 좌골 신경 C-섬유의 생체 내 단일 섬유 기록

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.