NIH (NS - 18205)에 의해 재정 지원.

1. 이점 레코딩을위한 물고기의 준비 <ol><li> 이전 절차를 시작하기 위해 포함 5 가지 주요 솔루션을 준비 : 목욕 솔루션, Tricaine (MS222), 포름 아미드, 신경 세포 내부 솔루션과 근육 내부 솔루션과 함께 목욕 솔루션 목욕 솔루션입니다. </li><li> 다음 72-96시간 세에서 Tricaine 욕조 솔루션의 장소 사이에 하나의 애벌레 zebrafish를 수집합니다. 마취에 노출 1 분 이내에 물고기 연락에 응답하지 않습니다. </li><li> 플라스틱 접시 위에 생선을 놓고, stereomicroscope를 사용하여, 더블 에지 면도날로 목을 베다. </li><li> sylgard와 코팅과 목욕 솔루션 가득한 유리 녹음 챔버에 생선을 전송합니다. notochord 통해 electrolytically 날카롭게 텅스텐 핀을 삽입하여 각 끝에 고정. </li><li> 좋은 포셉 한 켤레에 대한 적절한 그립을 제공할 것입니다 핀과 함께 물고기의 주동이의 끝 근처에 피부 플랩을 만듭니다. 좋은 포셉 한 쌍의와 주동이의 핀 근처 재밌하여 overlying 피부를 제거합니다. 서서히 꼬리 방향으로 껍질 피부를 제거합니다. </li><li> 녹음에 방해가있을 근육의 수축을 차단하는 3~5분에 대한 포름 아미드 욕조 솔루션 3 MLS와 생선을 취급. 일반 목욕 ​​솔루션 5 번 씻으십시오. 부드럽게 텅스텐 핀의 측면과 scrapping하여 5-6 세그먼트를 통해 근육의 표면 층의 일부를 제거합니다. </li><li> 전기 노이즈를 차폐하는 패러데이 케이지에 수직 현미경으로 생선을 전송합니다. 현미경은 오래 4X 거리에 40x 목적과 0.5의 확대 노력, 고정 무대를 갖추고 있습니다. 현미경은 높은 전력에 따라 신경 및 근육 사이의 준비를 이동하기 위해 전동 XY 번역 무대에 장착할 수 있습니다. 미분 간섭 대비 광학의 사용은 뉴런을 시각화하는 데 도움이됩니다. </li><li> 0.5 X 줌에서 overlying 근육을 제거하고 척수를 노출하기 위해 넓은 구멍 15 미크론 피펫을 사용합니다. 부정적인 압력이 3 CC 일회용 주사기와 근육 세포를 통해 적용됩니다 하나씩 제거됩니다. 이 절차는 골격근의 5-6 등의 세그먼트에 대해 반복됩니다. 같은 넓은 구멍 피펫도 대상 빠른 골격 근육을 얻기 위해 복부 근육의 상위 두 레이어를 제거하는 데 사용됩니다. </li><li> 이것은 이점 레코딩을위한 준비를 완료하고 현미경 줌 약 1.6x로 증가합니다. </li></ol> 2. 이점 캡 모터 신경 세포의 레코딩 및 대상 근육 세포 <ol><li> 청소 세그먼트는 캡 뉴런의 위치를​​ 스캔합니다. 척수의 각 헤미 세그먼트는 하나의 대형, 10 미크론의 직경, 캡 모터 신경 세포가 포함되어 있습니다. 이 티어드롭 모양 소마는 segmental 경계의 꼬리 가장 끝 부분에서 척수 두라 아래에 피상적으로 위치하고 있습니다. 캡 신경 세포는 녹음 선택하고 인접 꼬리 부분에서 복부 대상 근육은 무결성을 확인하는 검사입니다. </li><li> 두 패치 전극 녹음, 신경 세포 하나는 근육에 대한 잠깐 위치하고 있습니다. 상부 전극은 힘든 척추 두라 있으며, 약 2 미크론의 팁 오프닝의 침투에 대한 얕은 테이퍼가 있습니다. 낮은 전극은 골격근의 낮은 저항 전압 클램프 녹음 험한 테이퍼가 </li><li> 척추 두라에 침투 약 30 도의 각도에 신경 전극을 위치에 대한 하나를 선택 캡 신경 세포에 주동이의 반 세그먼트. 약 60mm의 수은의 부드러운 지속적으로 긍정적인 압력을 적용하는 동안 축 드라이브 조작하는 사람을 사용하여 전극 사전. 전극이 두라을 통해 나누기로 캡틴 신경 세포는 초점의 구획 정리가 필요 전극에서 재관류에 의해 갑작스런 수 있습니다. </li><li> 전극이 접촉하면 총액 신경 소마 긍정적인 압력이 출시와 gigaohm의 인감 즉각적인 형성에 일반적으로 결과입니다. 그러나, 봉인이 양식에 실패 경우에, 그것은 부드러운 부정적인 압력을 적용하는 데 필요한됩니다. 인감 형성 후, 전극 전위는 - 80mV로 조정하고 부정적인 압력의 응용 프로그램은 세포막을 파열이다. 캡 신경 세포가 140-180 메가 ohms의 입력 저항 및 행동 잠재력 해당 overshoots 40 millivolts이 특징입니다. 이것은 실천 가능성이 elicited 때까지 현재의 클램프 아래 depolarizing 단계를 점진적으로 증가 주사에 의해 테스트됩니다. </li><li> 다음 현미경은 높은 전력에서 XY 전동 번역기를 사용하여 복부 근육에 이전됩니다. 빠른 근육 세포는 대상 필드에서 선택됩니다. </li><li> 근육 전극이 출시되면, gigaohm 도장을 형성하고 긍정적인 압력을 받고 대상 근육 세포, 방향 고급입니다. 전극 전위는 나트륨 채널을 inactivate로 -50 millivolts 조정하고 부드러운 형편에서 세포막이 파열됩니다. 100-200 메가 ohms으로 저항하고 큰 용량성 과도 신호 항목의 갑작스런 등장에 놓습니다. </li><li> 테스트하려면실천 가능성이 elicited 때까지 이점 녹화, 모터 신경 전류가 점진적으로 큰 주사에 의해 depolarized 있습니다. 이 시점에서 endplate 전류는 근​​육에 elicited해야합니다. 실패없이 endplate 전류 1 헤르츠 결과를 motorneuron의 지속적인 자극. 자극 주파수 100 헤르츠로 증가로 endplate 흐름은 진폭의 변동 자극을 10 초 이내에 기능을 고갈을 받고있다. </li></ol> 3. 대표 결과 일반적으로 신경 세포의 녹화는 최대 한 시간 동안 안정하지만 근육 세포는 같은 직렬 저항의 증가로 반영 저하. 이 문제가 발생하면 실험 중 종료 또는 다른 근육 세포는 패치가 고정되어 있습니다. 모든 녹음 한 시간 이내에 완료해야합니다. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="그림 1" /> 그림 1. motorneuron 작업의 가능성 (AP, TOP)과 전류 관련된 근육 endplate (EPC, 하단)의 레코딩. 캡 신경 세포에 대한 행동 잠재력은 오버 슈트 40 뮤직 비디오와 EPC는 1 밀리초 이하 일정한 시간 지수 시간 코스를 따라 300 μsec과 부패 이내 상승해야한다. <table border="1"><tbody><tr><td> 이름 </td><td> 조리법 </td></tr><tr><td> 바스 솔루션 (MM)을 </td><td> 134 NaCl, 2.9 KCl, 2.1 CaCl 2, 1.2 MgCl 2, 10 포도당, 10 나 - HEPES, pH를 7.8 </td></tr><tr><td> Tricaine 욕조 솔루션 </td><td> 0.02 % tricaine을 포함 바스 솔루션 </td></tr><tr><td> 포름 아미드 욕조 솔루션 </td><td> 2M 포름 아미드를 포함하는 바스 솔루션 </td></tr><tr><td> 신경 세포 내부 솔루션 (MM)을 </td><td> 115 K - 글루 콘 산, 15 KCl, 2 MgCl 2, 10 K-HEPES 5 K - EGTA, 4 MG - ATP, pH를 7.2 </td></tr><tr><td> 근육 내부 솔루션 (MM)을 </td><td> 120 KCl, 10 K-HEPES, 5 BAPTA, 산도 7.4 </td></tr></tbody></table> 표 1. 솔루션을 제공합니다.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 주로 고주파 자극하는 동안 소포 자료 및 재활용의 과정을 연구 zebrafish 이점 레코딩 (웬과 브렘, 2005)를 사용하고 있습니다. 이 과정은 주요 구성 요소는 시냅스에 의한 포인트 돌연변이로 생긴 전형적인 시냅스 전달이 궁극적으로 운동성의 돌연변이에 방해됩니다. 예를 들어, postsynaptic 수용체 집계 (오노 외., 2001, 2002, 2004)와 presynaptic 송신기의 합성 (왕 외., 2008) 취소 조정 수영의 결과를 방해 변이. 이점 녹음 따라서 행동, 유전학 및 생리학을 연결하는 기능 결함의 원인을 정확히 파악할 수있는 정보를 제공합니다. 이제 더 사려 분별이있는 발기인을 사용하여 캡 모터 뉴런에 과도 표현에 의해 시냅스 전송을 기본 프로세스를 해부하다 수 있어야한다. 이런 방식으로 지배적인 부정적 또는 변이된 유전자가 특별히 이러한 뉴런의 표현과 모두 행동 및 electrophysiological 결과는 결정 수 있습니다. 전체 세포 구성에 의해 제공되는 추가적인 장점은 잠재적으로 이러한 clostridial 독소와 칼슘 버퍼로 소포 재활용을 변경할 수 에이전트와 캡 소마의 투석 수 있습니다. 소마와 근육 사이의 짧은 거리로 인해 보급이 레코딩의 시간 프레임 내에 잘 발생한다. 이 준비의 가능성은 도청으로 시작했습니다. 시냅스의 연령과 표면 위치에있는 물고기의 투명성은 또한 광학 도구의 사용 (; Fetcho와 히가시 2004 Fetcho와 오말리, 1997)을 용이하게합니다. 우리는 FM 염료와 같은 Synaptophluorin (리튬 외., 2003)로 유전자 지표를 사용하여 생활 생선에 엔도와 exocytosis의 측정에서 매우 성공적으로되었습니다. 또한, 형광 복합 독소가 활어 (아이 바네즈 - 탈론 외., 2004)의 이미징을위한 시냅스 단백질을 분류하는 데 사용할 수 있습니다. 이 준비의 단순 감안할 때 그것은 미래의 학습에 큰 약속을 보유하고 있습니다.

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
<td>&nbsp;</td>
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<td>0.002 x 3 inch tungsten rod</td>
<td>&nbsp;</td>
<td>A-M Systems Inc</td>
<td>715000</td>
<td>&nbsp;</td>
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<td>Sylgard 184 Elastomer</td>
<td>&nbsp;</td>
<td>Dow Corning Corp.</td>
<td>&nbsp;</td>
<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

애벌레 zebrafish는 척수 모터 신경 세포와 근육 대상에서 동시 패치 클램프 녹음을 허용하는 최초의 척추 모델 시스템을 나타냅니다. 이 두 세포 유형과 시각 형태 및 위치에 기초하여 단일 segmental 캡 모터 신경 세포를 구별하는 능력에 접근의 직접적인 결과이다. 이 동영상은 캡 모터 신경 세포와 목표 근육 세포뿐 아니라 각 세포 유형에서 레코딩을위한 방법론을 식별하는 데 사용되는 미세한 방법을 보여줍니다. 캡 모터 신경 세포 유형의 식별은 염료의 작성에 의해 또는 행동 잠재력 파형과 셀 입력 저항으로 biophysical 기능에 의해 확인됩니다. 일시간 여러 다른 대상 근육 세포에서 장기적인 레코딩을 허용에 대한 일상적으로 마지막으로 모터 신경 세포의 녹음. 모터 신경 세포의 발사 패턴을 통해 컨트롤 신경근육학 교차점에서 시냅스 전달의 주파수 의존의 측정이 가능합니다. 대형 quantal 크기 및 전체 셀 전압 클램프가 제공하는 낮은 잡음 때문에, 하나의 이벤트가 모든 근육에서 해결하실 수 있습니다. 이 기능은 릴리스 속성, 소포 재활용뿐만 아니라 우울증과 신경 촉진 등의 기본적인 시냅스 속성의 연구를 수 있습니다. 모터 뉴런은 보통 세포 전극에 의해 자극된 상태네 궁극적 생체내 준비 이클립스 이전 신경근육학 모델 시스템이 제공하는 장점은 연구와 근육이 전체 세포 패치 클램프에 비해 너무 큽니다. zebrafish 준비는 유전자 변형 라인, morpholino의 최저, 약리 개입 및 생체내 이미징에 포함하는 방법의 다양한 electrophysiological 분석을 결합 의무가 있습니다. 이러한 접근 방식은, zebrafish의 돌연변이 라인에 의해 제공 신경근육학 질병 모델의 증가와 결합, 인간 신경근육학 질환의 새로운 이해 문을 엽니다.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

애벌레 zebrafish는 척수 모터 신경 세포 및 대상 골격근에서 동시 패치 클램프 녹음을 허용하는 최초의 척추 모델 시스템을 나타냅니다. 이 동영상은 segmental 캡 모터 신경 세포와 목표 근육 세포뿐 아니라 각 세포 유형에서 레코딩을위한 방법론을 식별하는 데 사용되는 미세한 방법을 보여줍니다.

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.1K 조회수.  Oregon Health and Sciences University. 애벌레 zebrafish는 척수 모터 신경 세포 및 대상 골격근에서 동시 패치 클램프 녹음을 허용하는 최초의 척추 모델 시스템을 나타냅니다. 이 동영상은 segmental 캡 모터 신경 세포와 목표 근육 세포뿐 아니라 각 세포 유형에서 레코딩을위한 방법론을 식별하는 데 사용되는 미세한 방법을 보여줍니다.

비디오: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

어두운 적응 슬라이스 준비에 마우스 망막 뉴런에서 패치 클램프 레코딩

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

연구

신경과학

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

귀에 세포의 패치 클램프 녹음 절연 Zebrafish에서

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subSubmitochondrial 소포 막 패치 클램프 녹음을 공연&gt; ATPase의 소포의 준비와 기술

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

해마의 Organotypic 조각의 쌍으로 전체 셀 녹음

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

신경 근육 접합 : 측정 시냅스 크기, 공 초점 형광 현미경을 사용하여 시냅스 단백질 농도의 분열 및 변경

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

급성 뇌 조각에서 신경 세포의 미세 회로의 전기 생리 및 형태 학적 특성은 페어 패치 클램프 녹음을 사용하여

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

뇌 조각에서 전체 셀 패치 클램프 녹음

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...