This work is supported by a National Science Foundation award (1145581) to LWG.

주 : 시카고 일리노이 대학의 동물 관리 및 사용위원회에 의해 승인 된 전체 절차는 국립 보건원 (NIH)의 지침에 따라 실시 하였다. 1 솔루션 및 문화 미디어 준비 <ol><li> 최대 6 개월 -20 ° C에서 모든 솔루션을 유지합니다. 최대 3 개월 동안 4 ° C에서 배양 배지를 보관하십시오. </li><li> DMEM 100 ml의 ITSX (인슐린 트랜스페린 - 셀레늄 보충제)의 한 펜 연쇄상 구균의 수용액 1 ml에 혼합하여 배양 배지 100 ㎖를 준비합니다. </li><li> DMEM 250 ㎖, 100 MM의 염화칼슘이 2.5 ml의 50 mM의 EDTA의 2.5 ML을 혼합하여 효소 솔루션을 준비합니다. </li><li> DMEM의 225 ML, FBS의 25 ML, 알부민 625 ㎎, 625 mg을 트립신 억제제를 혼합하여 불 활성화 솔루션을 준비합니다. </li><li> 154 밀리미터의 NaCl, 5.6 밀리미터의 KCl, 5.0 mM의 HEPES, 3.6 mM의 NaHCO3를, 5.6 mM의 포도당의 로크의 솔루션을 준비합니다. NaOH로 7.3로 pH를 조정합니다. </li><li>50 ㎎ / ㎖의 폴리-D-라이신 (PDL) 솔루션을 준비합니다. 코트 커버 슬립을 1 ㎎ / ㎖의 최종 농도를 사용한다. </li><li> 140 mM의 NaCl을, 5 밀리미터의 KCl, 2 mM의 염화칼슘 2, 1 mM의 MgCl2를, 10 mM의 HEPES-NaOH를, 10 mM의 포도당의 세포 외 용액을 준비합니다. ~ 310 nmol의 / kg의 삼투압과 NaOH로 7.3로 pH를 조정합니다. </li><li> 50 mM의 NaCl을, 100 mM의 TEACl, 5 밀리미터의 KCl, 2 mM의 염화칼슘 2, 1 mM의 MgCl2를, 10 mM의 HEPES의 피펫 솔루션을 준비합니다. ~ 290 nmol의 / kg의 삼투압과 NaOH로 7.3으로 산도를 조정합니다. </li></ol> 2 부신 격리 <ol><li> 크고 작은 해부 가위와 집게 이쌍 한 쌍의 압력솥. 전체 과정 동안 장갑을 착용하고 얼음 양동이에 해부 트레이를 배치합니다. </li><li> 주 0-3 일에 찍은 마우스를 사용합니다. 잘린 다음에 가압 CO 2 탱크와 동물을 안락사. </li><li> cervi에서 척추 다음 강아지의 뒷면을 줄이기 위해 위의 작은 세트를 활용<html> 칼은 지역을 꼬리 지느러미합니다. 피부 아래 표면적으로 잘라 체강 내에 뚫지해야합니다. </li><li> 다음으로, 멀리 척추에서 껍질 피부가 근육은 척추의 명확한 전망이 노출되도록. 여기에서 자궁 경부 척수에서 transectional 컷을 한 후 척추의 측면을 따라 두 개의 평행 한 컷을합니다. </li><li> 동물의 본체를 들고 집게 일쌍으로 척추를 잡아 신장이 노출 될 때까지 척추를 벗겨 집게의 다른 쌍을 사용합니다. </li><li> 이 때, 신장의 위쪽에있는 부신을 시각화. 그런 다음 조심스럽게 로크의 솔루션으로 가득 원뿔 튜브에 각 동물에서이 샘을 배치합니다. 참고 : 때때로 땀샘은 집게에 충실 할 것이다. 이 경우, 부드럽게 집게로부터 로크의 용액에 땀샘의 제거에 도움이 다른 한 쌍의 집게를 사용한다. 땀샘 2 시간까지이 상태에서 유지 될 수있다. </li></ol>전자 &quot;&gt; 3. 조직 소화 <ol><li> 한편, 거품을 15 분 동안 5 % CO 2 + 95 % O 2 효소액 사용 전에. 평형 솔루션은 분홍빛 / 오렌지 색상에 밝은 핑크 색상에서 켜지는지 확인합니다. </li><li> 2,025 단위 / ㎖의 최종 농도 갓 버블 효소액 파파인 추가. 파파인으로 효소 용액에 37 ° C에서 40 ~ 60 분 동안 각 동물에서 동맥의 각 쌍을 품어. </li><li> 각 튜브에 불 활성화 솔루션의 75 %를 추가하고 또 다른 10 분 동안 37 ° C에서 배양. 이 배양은 파파인 효소의 활성을 불 활성화. </li><li> 불 활성화 용액으로 10 분 배양 후, 부드럽게 새로 레이블 튜브로 땀샘을 전송 한 후 샘은 문화 미디어 3 배 씻는다. </li></ol> 4 셀 해리와 도금 <ol><li> 세안 후, 배양 배지의 200 μL에이 글 랜드 위치하게 한 후, 부드럽게 분비를 적정하고다운 200 μL 피펫 피펫 팁을 통해 더 이상 용액 조직의 큰 조각이 없을 때까지. NOTE : 적정하면, 200 μL 피펫 팁으로부터 하방으로의 힘으로 튜브의 하단에 분비를 유지 조심스럽게 천천히 결합 조직. 느린 연속 스트로크를 사용하여 특정 수는 결코 빠르거나 갑작스러운 피펫 없습니다. </li><li> 450 μL까지 배양 배지의 전체 볼륨을 가지고 추가로 250 ㎕의 문화 매체를 추가합니다. </li><li> 플레이트 60 X 15mm 배양 접시에 배치 일곱 12mm PDL 미리 코팅 된 커버, 각각에이 세포 함유 용액의 60 μL. </li><li> 일단 도금, 세포 생존을위한 환경을 보장하기 위해 30 분 동안 5 % CO 2의 꾸준한 흐름 / 공급 37 ° C로 설정되고 유지되는 인큐베이터에 접시를 놓습니다. 배양 후, 부드럽게 배양 접시에 미리 예열 문화 매체의 5 mL를 넣고 24 시간 동안 인큐베이터에 다시 접시를 반환 세포 무관 이전D 녹음. 주 : 일반적으로, 세포가 최대 4 일 동안 전기 생리 레코딩에 사용될 수있다. </li></ol> (5) 셀 식별 및 기가 옴 인감 형성 <ol><li> 세포 외 용액에 12mm coverslip에 놓습니다. 전체 부신 셀 제조에 사용되기 때문에, 대뇌 피질의 세포 (작고) 셀을하는 chromaffin보다 디머와 문화에하는 chromaffin 세포 (갈색 / 베이지 색상과 완벽에 가까운 원형 형태로 일반적으로 매우 밝은 (그림 1)을 섞는다. </li><li> 프로그램 P-97 풀러를 사용하여 네 단계의 패치 피펫을 당깁니다. 다음 유리의 용량을 줄이고 데 도움이됩니다 녹은 왁스로 피펫 팁을 찍어은 피펫 팁의 안쪽에서 끝이 &quot;불어 멀리&quot;하는 폴란드어이 왁스를 발생. 참고 : 패치 피펫 솔루션 가득 할 때, 패치 피펫은 일반적으로 1 ~ 2 MΩ의 저항이있다. </li><li> 몇 m 내에서 세포에 가까운 패치 피펫 접근icrons. 세포 부착 된 구성을 달성하기 위해, 셀에 가까운 패치 피펫의 접근 중에 약간 변형 셀을 찾는다. GΩ 시일이 달성 될 때까지 부드러운 흡입을 적용합니다. </li></ol> 6 셀 첨부 용량 녹음 <ol><li> GΩ 시일이 계시되면 EPC-7 플러스 패치 - 클램프 증폭기의 사용을 통해 세포 부착을 녹음하고 두 상 아날로그 락 - 인 증폭기 (장비 첨부 표 참조). 잠금 증폭기에서 50 MV (RMS)의 진폭과 주파수 20kHz의 사인파를 적용합니다. </li><li> 1 밀리 초 시정 및 24dB의 종속 증폭기의 출력 필터를 설정합니다. 잠금 증폭기와 같은 부드러운 흡입 펄스에 의해 생성 된 일시적인 정전 용량 변경 사항은 패치 전도도 (재) 추적에 투영법으로 패치 용량 (임) 추적에서 나타나는 (그림 2)의 위상을 설정합니다. 참고 : 재에서 시스템의 교정에 대한 세부 정보를 참조하십시오(19) 전파 방해. </li><li> &quot;아래로&quot;단계 (그림 3)에 의해 일반적인 정전 용량의 변화를 확인합니다. 참고 : 고체 기록이 하나의 소포를 내면화 나타냅니다 많은 아래 단계를 &quot;계단&quot;형 닮은으로 볼 수 있습니다. 패치 프로세스는 동작 전류는 일반적으로 대부분의 세포에서 기록 될 수 있기 때문에 기계적 자극의 역할을한다. </li><li> 해당 파일 내에서 mm / 일 / 년 및 자신의 개인 번호 (XX) 등의 각 셀에 연결된 기록 : 개별 날짜 등의 폴더로 파일의 이름을 지정합니다. </li></ol>

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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

세포 부착 커패시턴스 측정이 성공적으로 고품질 레코딩 얻기 위해서는 몇 가지 중요한 단계가 필요 : 부신으로부터 제조 1) 실용적이고 건강한 세포; 커버 슬립의 2) PDL 코팅; 3) 기가 옴 씰 형성; 4) 시스템의 노이즈 레벨; 5) 위상 보정. 동물의 수술 한 잠재적 수 변형이 가장 적합 기용으로 수술 접근을 조정하고, 박리시 손상을 방지하는 것이다. 전기 생리 녹음을 위해 이용 ?...

시냅스 전달은 신경 전달 물질 - 함유 소포 시냅스 세포 외 유출에 의해 매개되고, 이러한 소포는 장기적으로 신경의 통신을 유지하기 위해 신경 말단 내의 지방 세포 내 이입 재활용을 거쳐야한다. 시냅스 소포 사이클 전체적으로 셀룰러 통신의 더 나은 이해를 향해 필수적인 토대 구성 분자 기계 이해, 뇌 시냅스 전달의 중요한 역할을 감안. 셀 모델 시스템 중 부신하는 chromaffin 세포는 시냅스 소포 재활용의 기초가되는 분자 기계에 관하여 가장 정확한 통찰력의 일부를 제공하고 있습니다. 세포 외 유출, 신경 전달 물질 방출의 마지막 단계는, 대단히 공부 부신하는 chromaffin 셀 1,2를 사용하여 조사되었다. 사실, 분비 과립의 형성, 타겟팅, 도킹 및 융합을 조율 분자 선수 대부분 기인 DIV의 애플리케이션에 확인 된하는 chromaffin 세포 하나에서 ERSE 기술. 더욱이, 세포 외 유출에 관여하는 단백질의 단일 기계 소포 해상도를 허용 할 수있는 기회를 제공하여, 셀하는 chromaffin 소포 융합의 3 문제를 해결하기 위해 강력한 모델 남아있다. 세포 부착 된 용량 측정은 첫째 세포 외 유출 세 동안 단일 소포 융합 해결에 사용되었다. ~ 60 nm의 직경이 입증 된만큼 작은 소포 세포 외 유출은 세포 부착 구성 4-7에서 패치 클램프 기술로 세포막 어드미턴스 측정에 의해 검출된다. 어드미턴스는 회로 또는 장치에 전류가 흐르도록하는 방법을 쉽게의 척도로서 정의된다; 이것은 임피던스의 역이다. 따라서, 어드미턴스의 측정은 막의 커패시턴스의 이해를 제공한다. 이는 세포막 소포 막에 혼입함으로써 달성된다; 이 법인은 표면의 변화를 보여준다지역 8. 각 융합 막 소포는 커패시턴스 9,10에서 단계적으로 증가시킨다. 또한,이 측정은 어드미턴스 막 컨덕턴스 및 이벤트 exocytotic 3 중 융합 기공 컨덕턴스를 제공한다. 이 기술은 세포 외 유출 동안 단일 소포 반응 속도를 식별에 고유 한 도구를 제공하고, 우리의 연구소는 최근 하나의 소포 11, 12의 엔도 사이토 시스를 감지하는이 개념을 적용하고있다. 우리의 특정 관심이 많은 셀 (13)과의 연결 단자 (14, 15)에서 시냅스 소포 엔도 시토 시스에 대한 주요 경로로 기본적인 가사 구성 요소로 간주하고있다 clathrin 매개 엔도 시토 시스 (CME)입니다. CME는하지만, 그 반응 속도로 인해 단일 ​​세포 내 이입 된 사건을 검사하는 경우에 기술적 인 제한으로 잘 이해되지 남아, 생물학적으로 중요한 것으로 알려져있다. 하는 chromaffin 세포와 신경 세포 일 사이 exocytic 메커니즘의 유사성을 감안할 때, 그것이 와줘하는 chromaffin 세포 분열 메커니즘 가능성이 뉴런에서 시냅스 소포 엔도 시토 시스에 적용 할 수 있음을 ausible. 세포 부착 된 용량 측정은 각각의 세포 내 이입 이벤트를 모니터링하고 대부분의 방법으로 해결할 수없는 핵분열 반응 속도를 분석하는 데 이용되어왔다. 우리의 세포 - 부착 기록에서, 20 kHz에서 사인파 들고 잠재적 겹쳐되며, 출력 전류는 증폭기 로크에 두 단계에서 다른 채널에서 하나의 채널과 멤브레인 커패시턴스 막 컨덕턴스로 분리 16 18. 멤브레인 컨덕턴스 및 커패시턴스의 변화로부터, 하나의 가능성 소포 핀치 오프 이전 원형질막에 내재화 소포 연결 관형 막 목에 대응 핵분열 기공의 동력학을 계산할 수있다. 종합적으로,이 기술은 우리에게 CME 동안 소포 분열의 규제 메커니즘을 조사 할 수있는 기회를 제공합니다.

<table><tbody><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899</td><td></td></tr><tr><td>DMEM</td><td></td><td>15066024</td><td>Keep out of UV</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Cover Glass</td><td>Carolina Biological</td><td>633029</td><td>12 mm</td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>100 ml</td></tr><tr><td>Insulin-Trans-Sel-X</td><td>Life Technologies</td><td>51500056</td><td>Only thaw on ICE!</td></tr><tr><td>Papain</td><td>Worthington</td><td>39S11614</td><td></td></tr><tr><td>EPC-7 plus patch amplifier</td><td>HEKA</td><td></td><td></td></tr><tr><td>BNC-2090 data acquisition board</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Igor data acquisition software</td><td>Wavemetrics</td><td></td><td></td></tr><tr><td>P-97 pipette puller</td><td>Sutter Instruments</td><td></td><td></td></tr><tr><td>Microforge</td><td>Scientific Instruments</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instruments</td><td>B150-110-10</td><td>Outer diameter: 1.5 mm&lt;br /&gt;
Inner diameter: 1.10 mm&lt;br/&gt;
Length: 10 cm</td></tr></tbody></table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

세포 생존 및 기가 옴 시일의 품질은 셀에 연결된 커패시턴스 레코딩의 품질을 결정하는데 중요하다. 따라서, 종래 전기 생리 레코딩, 및 일반적인 생존 세포로도 1에 도시되어 효과적이고 효율적인 세포 배양을 조달하는 것이 중요하다. 연습 시간은 고품질 기가 옴 시일을 달성하는데 도움이 될 것이다. 하나 명확 프로토콜 단계 5.3에 기재된 바와 같이 패치 피펫 셀 다가 세포 변형을...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

마우스 부신하는 chromaffin 세포에서 세포 부착 된 정전 용량 측정을위한 방법

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.8K 조회수.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

비디오: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

마우스 부신 Chromaffin 셀 절연

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

연구

생물학

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

패치 - 클램프 커패시턴스 측정 및 CA 2 +</sup망막 조각의 단일 신경 터미널에서> 영상

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

신경과학

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

마우스 망막 신경절 세포의 시냅스와 인공 컨덕턴스와 동적 클램프 구현

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

면역학 및 감염병학

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

분 비 소포 및 Exocytosis 삼투성 스트레스의 효과 모니터링

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

칼슘의 동시 측정을위한 인간의 심방 근육 세포의 분리<sup&gt; 2 +</sup&gt; 과도하고 막 전류

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

기본 교양된 Murine Enterochromaffin 셀의 전체 셀 전기 생리학

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

마우스 부신하는 chromaffin 세포에서 세포 부착 된 정전 용량 측정을위한 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스 부신 Chromaffin 셀 절연

패치 - 클램프 커패시턴스 측정 및 CA^{2 + 영상}

마우스 망막 신경절 세포의 시냅스와 인공 컨덕턴스와 동적 클램프 구현

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

분 비 소포 및 Exocytosis 삼투성 스트레스의 효과 모니터링

칼슘의 동시 측정을위한 인간의 심방 근육 세포의 분리

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

기본 교양된 Murine Enterochromaffin 셀의 전체 셀 전기 생리학

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Ca²⁺ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

마우스 부신하는 chromaffin 세포에서 세포 부착 된 정전 용량 측정을위한 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스 부신 Chromaffin 셀 절연

패치 - 클램프 커패시턴스 측정 및 CA 2 + 영상

마우스 망막 신경절 세포의 시냅스와 인공 컨덕턴스와 동적 클램프 구현

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

분 비 소포 및 Exocytosis 삼투성 스트레스의 효과 모니터링

칼슘의 동시 측정을위한 인간의 심방 근육 세포의 분리

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

기본 교양된 Murine Enterochromaffin 셀의 전체 셀 전기 생리학

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

패치 - 클램프 커패시턴스 측정 및 CA^{2 + 영상}

Ca²⁺ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리