NSF (0,854,551)에 의해 기금을 마련했습니다.

1. 사전 실험 준비 <ol><li> 다음과 같은 솔루션을 (작품에 대한 표 1 참조) 준비 : () 1리터의 NFR (일반 개구리 벨소리), (B) 100 ML 10 % 염분, (C) 100 ML CMF (CA 2 + / MG 2 + - 무료 솔루션 ), (D) 1 ML ITS (인슐린 - 트랜스페린 - 셀레늄, 시그마 I1884), (E) 100 ML L-15 배양액 (F) 10 ML HCG (인간 chorionic 생식샘 자극 호르몬 시그마 CG-10), (g)는 100 ML K + - 내부 솔루션, 솔루션 1의 삼투 강도가 그 증기 압력 osmometer (예를 들면 Wescor 모델 # 5100C)를 사용하여 약 260 mOsM되어 있는지 확인하기 위해 확인해야 Amphotericin B.에 대한 (H) 100 ML K +-내부 솔루션 . </li><li> (B) 솔루션을 소독 필터링 및 (c). 솔루션 (A) (B)하며 (C) 4 ° C. 3 개월까지 저장할 수 있습니다 25 게이지 주사기 바늘과 주사기 필터를 통해 동결 건조 분말이 들어있는 유리 병에 1 ML deionized H 2 O를 추가하여 솔루션 (D)를 준비합니다. 이 최종 해결책 &#39;C30 일 동안 4 ° C에 보관. </li><li> 비커 나 플라스크에있는 모든 구성 요소를 결합하여 층류 후드에 솔루션 (전자)를 준비합니다. 최종 솔루션을 소독하고 멸균 원심 분리기 튜브에 10 ML aliquots을 전송 필터링 할 수 있습니다. -20 ° C.에 저장 </li><li> 25 게이지 주사기 바늘과 주사기 필터를 통해 동결 건조 분말이 들어있는 유리 병에 H 2 0을 deionized 10 ML을 추가하여 HCG (솔루션 (F)) 준비합니다. 이 최종 솔루션은 30 일 동안 4 ° C에 저장할 수 있습니다. </li><li> 접착 시아 노 아세틸렌 접착제와 유리 파스퇴르 피펫의 끝 부분에 Minutien 핀 (26002-10 좋아, 과학 도구)에 의해 적어도 두 개의 microdissection 도구를 제조. 피펫 약 0.5 cm의 끝을 지났 확장하는 핀의 날카로운 끝을 허용합니다. </li><li> 준비 문화 이틀전 일 다음 절차 번식을 유도. 평면 스와의 여성과 검게 착색되어 눈에 잘 띄는, 붉은 배설강을 관찰하여 Xenopus의 번식 - 준비 쌍을 식별남성의 전면 발 rface. 이 탈출 할 수 있도록 시간 순 각 개구리 하나는은 그물과 싱크에 복부 측면을 누른 상태로 유지합니다. 등쪽의 림프 주머니의 그물과 피하에 하나를 통해 솔루션 1 ML (F)를 주입. </li><li> 물을 대상 십갤런 탱크에서 함께 번식 쌍을 배치합니다. 동물 새로 마련 및 수정 된 알을 짓밟되지 않도록하려면 약의 메쉬 크기 ½ 탱크의 바닥 (그림 1A) 위의 &quot;장착되어 약 1-2인치와 스크린 층을 설치합니다. </li><li> 동물 amplexus에 있고 수정 된 계란이 화면 아래에있는 층 (그림 1b)에 관찰 될 때까지 12-48 시간에 방해받지 개구리 둡니다. </li><li> 탱크에서 동물을 제거하지만, 더 이상 최소 24 시간의 알은 그대로 두십시오. 개구리의이 한 쌍의 6 주 후에 다시 사육 할 수 있습니다. </li><li> 탱크의 바닥에서 배아를 느슨하게 4, 5 60x15 mm의 컬트로 전송10 %의 생리 (솔루션 B)를 포함 우레 요리. Niewkoop와 페이버 (ref. 20)의 계획에 따라 단계로 배아를 정렬하십시오. 유용한 배아 무대 22-24에 해당 될 것입니다. 그림 2B는 (약 무대 28) 유용 할 개발에 따라 너무 멀리있는 배아를 표시 할 경우 그림 2A는 약 스테이지 22 배아를 보여줍니다. 그것은 건강한 배아를 선택하는 것이 중요합니다 : 빛 갈색과 흰색 mottling과 외관에 부드러운되어있는이 이상적입니다. 큰 검은 색 또는 흰색 패치가 태아는 일반적으로 건강에 해로운되어 사용할 수 있습니다. </li></ol> 2. Xenopus의 태아의 Microdissection <ol><li> 층류 흐름 후드, 라벨 내부와 약 절반 세 60x15 mm 멸균 문화 10 % 식염수에 요리 기입하고, CMF과 하나가 있습니다. </li><li> 10 %의 염분이 포함 된 요리 중 하나에 살균, 유리 파스퇴르 피펫 전송 5-10 단계 22-24 배아를 사용합니다. 스테레오 줌의 도움이 해부와후드 내부 ING 현미경 (10 × 접안 렌즈와 0.6-5 배)는 무균 # 5 포셉 (11251-30 좋아, 과학 도구)의 두 쌍을 사용하여 각 배아에서 젤리 코트와 vitelline 막을 제거합니다. (인물 3A 및 3B를 참조하십시오.) </li><li> 10 % 식염수의 나머지 2 요리를 통해 마지막으로 CMF를 포함하는 요리로, 한 번에 하나의 그들에게 전달하여 맨 배아를 씻으십시오. 각 전송을 위해 새로운 멸균 피펫을 사용하고 요리에서 요리에 전송 솔루션의 볼륨을 최소화합니다. </li><li> 회전 단계 1.5에서 만들었 microdissection 도구를 사용하여, 포셉 한 쌍의 완만하지만 단단히 각각의 배아를 개최하고, 신경 튜브과 동물의 가장 등쪽 부분에 위치하고 있습니다 연관된 myotomes를 제​​거합니다. 세 조각, 신경 튜브의 위치와 (그림 4A) 있도록 외부 단지 복부의 양쪽 끝에 한을 만들어이 작업을 수행합니다. 거리에 난황 granul에서 요리의 깨끗한 부분으로 각 해부 신경 튜브 / myotome 이동에스와 다른 부스러기 (그림 4B). 등쪽 - 복부 축, 그리고 신경 튜브와 myotomes의 위치는 그림 4에 표시됩니다. </li><li> 이 솔루션에서 15 분 정도 후 (CMF) 사용 포셉 해부 조직의 무료 색소 피부를 리프트 및 삭제합니다. 그들은 서로 (그림 4C)에서 dissociated 될 때 추가로 30-60 분 후, 세포는 &quot;모래 더미&quot;를 형성합니다. </li></ol> 3. 신경 근육 공동 문화의 작성 <ol><li> 문화 매체의 10 ML 튜브를 해동하고 무균 70 μl를 추가 ITS 35 NG / ML BDNF (시그마 B3795). </li><li> 라벨 및 멸균 35mm 문화 요리 (FD35-100 세계 정밀 계측기) 약 반 L-15 배양액 (솔루션 (E)), 해부하는 각 배아에 대한 하나를 채 웁니다. </li><li> 불꽃 위에 테이퍼 부분을 누른 상태 유리 파스퇴르 피펫의 각 끝을 쥐었고하여 도금 피펫을 제조. 에 대한 1로 끝을 분리 당겨0cm 후 약 0.2 mm의 팁을 얻을 수 있도록 끝을 중지. </li><li> 그린 솔루션의 양을 최소화 한 배아에서 &quot;모래 더미를&quot;빨아 도금 피펫을 사용합니다. 배양 접시의 바닥에 세포를 배출. 여러 줄에 플레이트 세포를. 도금 undifferentiated 세포를 (그림 5A 및 5B) 관찰. </li><li> 셀 시간이 접시에 부착 할 수 있도록 적어도 15 분 동안 방해받지 도금 세포의 요리를 둡니다. </li></ol> 4. 패치 클램핑 신경 근육 시냅스 <ol><li> 근육 세포가 스핀들 모양이되어 척추 뉴런이 긴 프로세스 (그림 6) 연장 : 문화에 12-24 시간 후, 도금 전지는 별개의 형태학의 특성에 걸릴. neurite varicosities와 근육 세포 사이의 시냅스 기능 연락처를 동시 페어링 electrophysiological 레코딩을 확인 할 수 있습니다. &quot;M&quot;, &quot;S&quot;와 &quot;V&quot;는 neuronal, 근육 세포를 각각 참조소마와 presynaptic varicosity. </li><li> presynaptic varicosity 한과 근육 세포에 대해 하나 : 기록을위한 두 패치 전극을 준비합니다. </li><li> presynaptic 전극 들어, 다음과 같이 amphotericin 함유 솔루션을 준비 : 5 밀리그램 amphotericin B (시그마 A4888)를 포함하는 1.5 ML의 microcentrifuge 관에 100 μl DMSO를 추가합니다. 10 초에 소용돌이이 솔루션입니다. 다음으로, Amphotericin B (솔루션 (H))에 대한 + - 내부 솔루션 625 μl K가 포함 된 두 번째 microcentrifuge 튜브에이 솔루션을 10 μl를 추가합니다. 위와 같이 와동. </li><li> amphotericin 함유 K 단계 4.3에서 조리 + - 내부 솔루션과 K +-내부 솔루션 (솔루션 (g))과 후 신경 전극과 미리 신경 전극을 입력합니다. </li><li> NFR과 배양 접시에서 미디어를 교체하고 위상 대비 광학가 장착 역 현미경으로 변경합니다. </li><li> 단지 각각의 목표 위의 두 전극을 배치합니다. 전체 셀 공동을 확보varicosity 전극으로 구멍 패치 구성하기 전에 근육 세포 전극과 nfiguration. </li><li> 시냅스의 기능을 확인하려면, 소프트웨어 제어하에 (예 : Axopatch 200B, 분자 장치) 패치 클램프 증폭기로 varicosity을 depolarize (pCLAMP 9, 분자 장치의 예)와 동시 presynaptic과 postsynaptic 전류를 관찰합니다. 그림 7은 본 전류를 보여줍니다 40 뮤직 비디오에 -30 뮤직 비디오에서 10 뮤직 비디오 단위의 presynaptic varicosity에게 전압의 단계를 depolarizing에 대응 인치 presynaptic 셀에 볼 안쪽으로 전류가 그렇단 + 및 CA 2 +와 K +를하여 외부 전류에 의해 실행됩니다. postsynaptic 셀에 기록 된 전류는 varicosity에서 출시 된 신경 전달 물질에 반응하고 nicotinic 아세틸 콜린 수용체 채널을 통해 오세영의 +에 의해 주로 수행하고 있습니다. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+neurotrophins+and+schwann+cell-derived+signals+on+neuronal+survival/growth+and+synaptogenesis.">Differential effects of neurotrophins and schwann cell-derived signals on neuronal survival/growth and synaptogenesis.</a> J. Neurosci. 23 (12), 5050-5060 (2003).</li><li>Dan, Y., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hebbian+depression+of+isolated+neuromuscular+synapses+in+vitro.">Hebbian depression of isolated neuromuscular synapses in vitro.</a> Science. 12 (5063), 1570-1573 (1992).</li><li>Xiao, Q., Xu, L., Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+regulation+of+neurotransmitter+specification+and+embryonic+neuronal+calcium+spike+activity.">Target-dependent regulation of neurotransmitter specification and embryonic neuronal calcium spike activity.</a> J. Neurosci. 30 (16), 5792-5801 (2010).</li><li>Niewkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin). , (1967).</li></ol>

관심 없음 충돌이 선언 없습니다.

motoneurons 및 근육 세포의 성공적인 공동 배양의 주요 단계를 적절하게 Xenopus 개구리 유도 사육에서 생산 배아를 조작의 사용 및 undifferentiated 척추 뉴런과 myoblasts의주의 무균 해부 있습니다. 빨리 자주 개발을 halts 이동로가 약 정도 (10) 개최에 도달 할 때까지 수정 된 달걀은 그대로 남아있을 것입니다. 건강한 태아는 부드러운 모양과 갈색과 흰색 얼룩덜룩 한 색소에 의해 식별됩니다. 이 근세포가 크게 차별화 한 단지 신경 튜브 닫히면 후에 전에 개발 중에 지점이기 때문에 스테이지 22-24 태아가 가장 유용합니다. 또한, 이전 배아의 세포는 CMF에 잘 해리하지. 그 배에 강력하게 준수로 vitelline 막 제거 할 때주의가 필요합니다. 배아는 그대로 나옵니다 있도록 막은 날카로운 집게를 사용하여 해체해야합니다. 또 다른 중요한 precaution은 배양 접시 (오히려이 정착 두는 것보다)의 하단에 세포를주의 깊게을 배치하는 것입니다. 이 세포가 배양 접시를 준수 될 확률이 높아집니다으로이 방법을 사용하는 것이 좋습니다. 신경과 근육 사이의 기능 neurotransmission 단 postsynaptic 기록으로 결정 할 수 있으며, 자주 innervation 후 자발적인 근육 수축의 관찰에 의해 ascertained 수 있습니다. 취소 innervated 근육 세포는 문화에 경련하지 않습니다. 혼자 Postsynaptic 녹음이 미니어처 endplate 전류 또는 전위하지만 evoked 출시 측정 사전 및 postsynaptic 전류 presynaptic 자극, 그리고 상관 관계 패치 - 클램프 더블을 필요로 필요를 기록하는 데 유용합니다. 여기에 설명 된 페어 - 패치 녹화 방법 외에,이 준비는이 수있는 neuronal 소마 5 삼분의 일 피펫을 소개 할 수있는 기회를 제공하는 행위 가능성이 THA의 생성t는 presynaptic varicosity에 전파하고 신경 전달 물질의 방출을 유도 할 수 있습니다. 또한, 중재 할 생각이 신경 전달을 조절 아르 putative 대리인은 시냅스의 양쪽에 도입 될 수있다 : 근육 세포 피펫을 통해 또는 소마에 배치 삼분의 일 피펫의 확산을 통해.

<table border="1"><tbody><tr><td> 장비 / 용품 </td><td> 공급 업체 </td><td> 카탈로그 / 모델 번호 </td><td></td></tr><tr><td> 인슐린 트랜스페린 - 셀레늄 </td><td> 시그마 </td><td> I1884 </td><td></td></tr><tr><td> 인간 Chorionic 생식샘 자극 호르몬 </td><td> 시그마 </td><td> CG-10 </td><td></td></tr><tr><td> 증기 압력 Osmometer </td><td> Wescor </td><td> 5100C </td><td></td></tr><tr><td> Minutien 핀 </td><td> 정밀 과학 도구 </td><td> 26002-10 </td><td></td></tr><tr><td> Amphotericin B </td><td> 시그마 </td><td> A4888 </td><td></td></tr><tr><td> 집게 </td><td> 정밀 과학 도구 </td><td> 11251-30 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 솔루션 이름 </td><td> 조리법 </td></tr><tr><td> (A) NFR (일반 개구리 벨소리 (MM)을 </td><td> 116 NaCl, 2 KCl, 1.8 CaCl 2, 5 오세영 + - HEPES, pH를 7.35. </td></tr><tr><td> (B) 10 % 염분 </td><td> 10 ML NFR 90 ML은 H 2 O를 deionized </td></tr><tr><td> (주) CMF (CA 2 + / MG 2 + 무료 솔루션) </td><td> 125 NaCl, 2 KCl, 1.2 EDTA (에틸렌 다이아 민 테트라 초산), 5 오세영 + - HEPES, pH를 7.35. </td></tr><tr><td> (E) L-15 문화 미디어 </td><td> 50 ML L-15 미디어 (시그마 L1518) + 50 ML의 NFR. </td></tr><tr><td> (g) K +-내부 솔루션 (MM)을 </td><td> 116 KCl, 1 NaCl, 1 MgCl 2, 10 EGTA, 5 HEPES, pH를 7.3 </td></tr><tr><td> Amphotericin B에 대한 (H) K +-내부 솔루션 (MM)을 </td><td> 52 K 2 SO 4, 38 KCl, 1 EGTA, 5 HEPES, pH를 7.3 </td></tr></tbody></table></td></tr></tbody></table> TablE 1.

simultaneous pre- and post-synaptic electrophysiological recording from xenopus nerve-muscle co-cultures

신경 전달 물질의 방출과 presynaptic 이온 전류의 커플 링에 대한 많은 정보는 특히 invertebrate 준비, 오징어 거대한 시냅스 1 일부터 취득했습니다. 그러나, 준비를 제외하고 여기에 설명 된, 몇 가지 척추의 준비는 신경 전달 물질 자료와 presynaptic 이온 전류의 동시 측정을 할 수 있습니다있는 존재합니다. (때까지 12-24시간 내에 기능 시냅스를 형성하고, 며칠 동안 신경과 근육 세포 개발과 신경의 상호 작용을 연구하는 데 사용할 수있는, 배아 Xenopus의 motoneurons와 근육 세포는 실온에서 간단한 문화 매체에서 함께 성장하실 수 있습니다 전면에 자라 난)가 발생합니다. 다른 척추 동물의 준비 이상이 공동 문화의 장점은 준비의 단순함, 상온에서 문화와 작업을 유지 할 수있는 능력, 2를 형성 시냅스의 준비 접근성을 포함-4. 준비는 presynaptic 이온 채널과 송신기 릴리스 5-8의 규정에의 biophysical 속성을 공부하고 널리 사용되었습니다. 또한, 준비는 9-12 neurite 가지와 synaptogenesis 연구, 신경 전달 물질 릴리스 13-15, neuromodulation 16,17의 diffusible 메신저의 역할의 분자 메커니즘, 그리고 체외 신경 소성 18 등 다른 용도로 자신을 빌려하고 있습니다 - 19.

그림 4B는 즉시 22 Xenopus의 배아. 그림 4C 그, CA 2 +-MG 2 + 무료 솔루션 피부 제거 및 부화 (CMF, 솔루션 후 (표시하는 단계에서의 제거 후 코드 / myotome 격리 된 척추의 등보기를 보여줍니다 C)), 셀은 &quot;모래 더미&quot;로 해리와 도금을위한 준비가되어 있습니다. 문화 매체 세포에 도금 약간의 형태학의 변화 (그림 5B)를 전시하지만, 문화에 이십사시간 후 독특한 모양에 걸릴 즉시. 뉴런이 확장 neurites하면서 구형 유지하면서 근육 세포는 스핀들 모양이 될 근육 세포와 시냅스 (그림 6)에서 정교한 varicosites.주세요 presynaptic varicosity과 postsynaptic 근육 세포 (그림 7)에서 동시 페어링 패치 녹음이 신경 전달 물질의 방출과 resulta과 관련된 presynaptic 안쪽과 바깥쪽으로 전류를 보여NT postsynaptic 전류를 흥분성의. <img src="/files/ftp_upload/50253/50253fig1.jpg" alt="그림 1"/> 그림 1 A :.. 화면 꼭대기에 결합 양동이의 개구리 사육 쌍 B : 수정 된 계란과 amplexus에 개구리. <img src="/files/ftp_upload/50253/50253fig2.jpg" alt="그림 2"/> 그림 2 A :.. &quot;적합&quot;단계 22 배아 B : &quot;적합&quot;단계 28 배아. 스케일 바는 1mm를 나타냅니다. <img src="/files/ftp_upload/50253/50253fig3.jpg" alt="그림 3"/> 그림 3 :. 스테이지 22 배아 vitelline 막과 젤리 코트의 제거 전에. 화살표는 젤리 코트의 바깥 쪽 가장자리로 가리 킵니다. vitelline 막이 embr에 밀접하게 준수야와 거의 보이지 않는 B는 :. 맨 배아. 스케일 바는 1mm를 나타냅니다. <img src="/files/ftp_upload/50253/50253fig4.jpg" alt="그림 4"/> 그림 4 A : 될 수있는 해부하는 부분 B를 나타내는 점선이있는 스테이지 22 배아 :.. 배의 심하게 격리 등쪽 부분을, &quot;D&quot;와 &quot;V&quot;는 배아의 등 및 복부 측면을 참조하는 동안 &quot;m&quot; 와 &quot;N&quot;은 mytomes과 신경 튜브의 대략적인 위치를 나타냅니다 C는 :. CMF에서 60 분 후 세포의 &quot;모래 더미&quot;. 스케일 바는 1 음, 그리고 B와 C에 대한 0.5 mm를 나타냅니다. <img src="/files/ftp_upload/50253/50253fig5.jpg" alt="그림 5"/> 그림 5 A :. 바로 도금 후 문화에 세포의 5 배 전력 전망이 있습니다. B : 40x에서 같은 문화. 규모바 40 μm 및 B. 5 μm을 나타냅니다 <img src="/files/ftp_upload/50253/50253fig6.jpg" alt="그림 6"/> 그림 6. neuronal 소마 (S), presynaptic varicosity (V) 및 postsynaptic 근육 세포 (M)의 식별과 문화에 신경 근육 접합부. 규모 바 : 5 μm. <img src="/files/ftp_upload/50253/50253fig7.jpg" alt="그림 7" fo:content-width="5in" fo:src="/files/ftp_upload/50253/50253fig7highres.jpg"/> 10 MV 증분 전압의 응용 프로그램에 대한 응답으로 보는 그림 7. Presynaptic (위)와 postsynaptic (하단) 전류가 -30 뮤직 비디오에서 40 뮤직 비디오에 presynaptic varicosity에 제출 단계를 반복합니다. 전압 단계는 presynaptic 흔적의 일부 옆에 표시됩니다. 두 세포의 개최 가능성은 -70 MV했다.

Watch this Scientific Journal Video about Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures at JoVE.com

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

이 동영상은 배아의 주 문화를 성장하는 데 사용되는 절차를 설명합니다<em&gt; Xenopus</em&gt; 신경과 근육 세포와 동시 사전 및 사후 신경 패치 클램프 녹음을 만들기위한이 준비 유용성.

동시 사전 및 사후 신경 Electrophysiological 녹화에서<em&gt; Xenopus</em&gt; 신경 근육 공동 문화

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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Neuroscience

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

10.9K 조회수.  David Geffen School of Medicine at UCLA. 이 동영상은 배아의 주 문화를 성장하는 데 사용되는 절차를 설명합니다 Xenopus 신경과 근육 세포와 동시 사전 및 사후 신경 패치 클램프 녹음을 만들기위한이 준비 유용성.

비디오: Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

의 전극 제작 및 주입 Aplysia californica 손상, 자유롭게 행동 동물의 다중 채널 및 신경 근육질 레코딩을위한

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

연구

신경과학

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oocytes의 표현 이온 채널 패치 클램프 녹음

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

생물학

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

성인 마우스에서의 모터 단위 제작 룸버 Motoneuron과 포스의 동시 세포 녹화<em&gt; 생체에</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

에 Electrophysiological 녹음 Drosophila 엠브료

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

준비 및 Xenopus laevis Tectal 신경의 전체 셀 패치 클램프 기록에 대 한 프로토콜

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

제노푸스 난모세포로부터의 갭 접합 전류 기록

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

Put a Petri dish with a coated microelectrode array inside under a stereomicroscope. Bring the array into focus, and center a six-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add eight microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber.
Place the chamber inside an incubator at 37 degrees Celsius for about an hour. After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused. Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue, covering the groove.
Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges. Put the multi-electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize, and then, record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes. Repeat the recordings a total of two times.
To ensure stable extracellular conditions, exchange the extracellular solution after every recording session. If desired, disinhibit the network by applying extracellular solution containing one micromolar of strychnine and 10 micromolar of gabazine and wait for at least two minutes before recording the electrical activity.

동시 사전 및 사후 신경 Electrophysiological 녹화에서

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