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 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>
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	</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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Neuroscience

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

6.8K 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 ...

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.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 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 ...

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.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

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.

조류 배아 Vestibulocochlear 신경 청각 섬유의 선택적 추적

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.

온전한 성인 Zebrafish의의 뇌에 전기 생리학 기록

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.

<em&gt; 체외</em근육의 구 심성의 소성 특성을 연구&gt; 성인 마우스 근육 - 신경 준비

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.

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 ...

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.

심각한<em&gt; In Vivo</em&gt; 마취 된 래트에서의 Hyperdirect 경로로부터의 국소 장 전위 및 다중 단위 활성의 전기 생리 학적 기록

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.

자유롭게 수영 물고기에서 이동 식 Tetrodes에 의해 뉴런의 무선 전기 생리적 기록

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