Name: whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins
Uploaded: 2017-05-22T14:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

우수한 기술 지원을 위해 Maila Reh, Tharsana Tharmalingam, 특히 Altina Klein에게 감사드립니다. 이 연구는 독일 연구 재단 (DFG) (SFB1078 B2, FOR1279 SPP1665 to PH)과 Catalysis, UniCat, BIG-NSE (JV) 및 E4 (PH)의 우수 통합 클러스터 개념에 의해 지원되었습니다.

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The authors have nothing to disclose.

정의 된 이온 및 전기 조건에서 역전 전위의 결정은 ChR의 가벼운 활성화 후에 운반 된 이온 종에 대한 정보를 제공합니다. 독점적으로 하나의 이온 종이 복잡한 생리 학적 매질에서 변화되고 얻어진 역전 전위가 이론상의 Nernst 전위에 따라 이동하면,이 이온 종이 유일한 수송 된 것입니다. 그러나 ChR의 경우 전도 이온 간의 경쟁은 물론 다른 이온 종에 대한 침투성 때문에 Nernst 방정식에서 예상했던 것보다 반전 전위 이동이 일반적으로 덜 명확합니다. 이 경우 상황이 더욱 복잡해지고 잠재적으로 수행되는 모든 이온을 다양한 측정 솔루션을 필요로하는 별도로 교환해야합니다. 또한, 대부분의 CCR 및 제 1 인공 ACR ( 9 , 10 , 21) 은 양성자에 대해서는 전도성이지만, 양성자 농도양성자 농도는 역전 전위를 이동시킬뿐만 아니라 수소 결합 네트워크의 변화, 이온 결합 상태의 변화로 인해 이온 전도도 9 , 광전류 운동 21 , 36 및 분광 광 민감도 11 , 37 에도 영향을 줄 수 있으므로 특히 철저한 분석이 필요하다. 개별 잔기 및 2 차 구조 변화. 양성자 전도성은 위에서 언급 한 단백질 변화를 피하기 위해 pH를 일정하게 유지하면서 Na + , Ca 2+ 또는 Cl - 와 같은 잠재적으로 전도 된 다른 모든 이온의 농도를 줄임으로써 해결할 수 있습니다. 비 전도성 대용 물은 일반적으로 양이온 12의 경우 NMG + , 음이온의 경우 글루코 네이트, 2- ( N- 모르 폴리 노) 에탄 설포 네이트 (MES - ) 또는 아스 파르 테이트와 같이 부피가 크고 대전 된 분자입니다. 다른 i에서 전위 역전 비교onic solution은 Goldman-Hodgkin-Katz 전압 방정식에 의해 평형 조건에서 상대 이온 투과성의 계산을 허용한다. 그러나 평형 조건을 넘어선 이온 경쟁을 고려하여 순 광전류에 대한 다양한 이온의 정확한 기여를 정량화하기 위해서는 복잡한 수학적 모델이 필요하다. ChR에 의해 운반되는 이온의 조성을 아는 것은 특히 신경 생리 학적 맥락에서 ChR 적용으로 인한 부작용을 밝혀내는 데 도움이됩니다. 예를 들어, 장기간 ChR 활성화는 세포 내 산성화를 유발할 수 있거나 또는 수행 된 칼슘 2+ 는 시냅스 방출과 이차 전달 경로를 유발할 수있다. 세포는 일반적으로 조직 내에 단단히 싸여 있기 때문에 미생물 로돕 신의 활성화는 세포 내 이온 조성뿐만 아니라 세포 외 관강에도 영향을 미쳐 n에 영향을 미친다주변 셀 41 . 이온 선택성에도 불구하고, 광전류 진폭, 불활 화, 불투명, 광 감도 및 운동 특성과 같은 ChR의 다른 생물 물리학 적 특징은 종종 광 생성 기술을 포함하는 실험을 수행하고, 설계하고 해석하는 데 관심이있다. 이러한 속성 중 일부는 위의 프로토콜 18 , 37 , 42에 설명 된 프로토콜에서 확인할 수도 있습니다. ChR 발현 세포의 빛 감도를 조사하기 위해, 빛의 세기는 광원의 힘을 조절하거나 중립 밀도 필터로 활성 광선을 감쇠시킴으로써 변화 될 수있다. 분광 감도를 얻으려면 다색광 광원이 필요하며 강도는 모든 파장에 대해 동일한 광자 유속 및 대역폭으로 조정되어야합니다. 고강도 스펙트럼은 짧은 광 펄스 또는 광 감응 기의 경우 광 수용체의 흡수 스펙트럼과 유사합니다.레이저 플래시가 사용됩니다. 그렇지 않으면 적응 현상과 광 화학적 역행이 발생할 수 있습니다. 완전히 비활성화 된 ChR (전체 photocycle 회전 시간)에서 부분적으로 비활성화 된 상태에서의 복구는 두 개의 광 펄스 사이에 어두운 간격이 증가하는 이중 펄스 프로토콜을 적용하여 조사 할 수 있습니다. 위에 논의 된 ChR의 생물 물리학 적 특성은 실험적 요구 사항에 적합한 ChR을 선택하는 데 중요합니다. 전기 생리 학적 기법을 통해 이러한 생물 물리학 적 특성을 도출하는 것은 분광학 방법으로는 거의 다루지 않는 단백질 기능과 직접적으로 유사합니다. 전압 클램프 측정을 성공적으로 수행 한 후에는이 기술을 부위 지정 돌연변이 유발 9 , 11 , 43 , 44 , 헬릭스 - 셔플 링 37 , <supclass = &quot;xref&quot;&gt; 45, 표적 서열 46 의 도입 또는 다른 단백질과의 융합 41 , 47 . 이것은 ChR과 그 작동 방식에 대한 분자 지식을 증가 시켰고 변화된 동역학, 전도도, 분광 감도 및 이온 선택성을 가진 ChR 변이체의 높은 다양성을 창출했다.

Channelrhodopsins (ChR)는 운동성 녹조류의 눈 지점에서 발생하는 광 게이 티드 이온 채널이며 광 택 및 공포 반응에 대한 기본 광 센서 역할을합니다 1 . ChR은 2002 년에 처음으로 기술 된 이래로 optogenetics의 새로운 분야에 대한 길을 열었고 골격근, 심장 또는 뇌의 다양한 흥분성 세포, 예를 들어 3 , 4 , 5 에 적용될 수 있습니다. 표적 세포에서 ChR을 발현 시키면 각 세포의 가벼운 조절 가능한 이온 투과성을 갖게된다. 연결 컨텍스트에서 이것은 활동 전위 (AP) 발화의 활성화 6 , 7 , 8 또는 억제 9,10 (수행 된 이온에 따라 다름)을 공간적 및 시간적ChR 변이체의 이온 선택성이 어떻게 그것의 광 생성 적용을 결정 하는지를 강조하는 빛의 정밀도. Chlamydomonas reinhardtii 와 Volvox carteri에서 처음으로 발견 된 ChR은 양성자뿐만 아니라 나트륨, 칼륨과 같은 1가 양이온, 칼슘과 마그네슘과 같은 2가 양이온에 덜 영향을 미친다 11,12,13. 오늘날, 70 가지 이상의 천연 양이온 전도성 채널 로돕신 (CCRs) 14 , 15 , 16 , 17 및 광전류 크기, 분광 감도, 동역학 및 양이온 선택도를 이용할 수 있습니다. 신경 과학에서 CCR은 a다시 세포를 활성화하고 AP를 트리거하는 데 사용되는 경우, 빛에 의해 구동되는 미생물 펌프는 수년간 뉴런을 조용하게하는 유일한 길항제였다. 2014 년에 두 그룹은 동시에 CCR이 분자 공학을 통해 추정 이온 전도 기공을 따라 극성을 바꾸어 음이온 전도성 채널로도 틴 (ACR)으로 전환 될 수 있음을 보여주었습니다 9 , 21 . 그 결과, 여러 가지 cryptophyte alga 22 , 23 , 24 에서 자연 ACR이 확인되었다. 가장 중요한 것은, ACR의 가벼운 활성화는 흡수 된 광자 당 단 하나의 전하만을 운반하는 미생물 펌프보다 훨씬 낮은 광 강도에서 신경 세포 활동의 억제를 허용하는 성인 뉴런에서 염화물 전류를 매개한다는 것이다. ChR 활동은 HEK293 세포의 빛 유도 전류의 전기 생체 학적 패치 클램프 기록으로 직접 해결할 수 있습니다. 패치 클램프기술은 원래 1970 년대 후반에 개발되었고, Hamill et al. 높은 전류 분해능과 멤브레인 전압의 직접 제어로 작은 셀 (전체 셀 모드)에서 전류 엔티티를 기록 할 수 있습니다. 세포 배양에 적용된이 기술은 이온 및 전기 기록 조건을 정확하게 제어하고 총 전류에 대한 이온의 상대적 기여와 함께 이온 선택성을 연구 할 수있게합니다. 여기서 우리는 염소 이온 전도도를 증명하기 위해 다양한 세포 외 염화물 농도 하에서 전류 - 전압 관계를 기록함으로써 Proteomonas sulcata ( Ps ACR1) 22 , 23 의 음이온 전도성 채널로도 펩신에 대한 이온 선택성의 시험을 예시한다.

<table><tbody><tr><td>HEK293 cells</td><td>Sigma Aldrich</td><td>85120602</td><td>Human embryonic kidney cells</td></tr><tr><td>Retinal</td><td>Sigma Aldrich</td><td>R2500</td><td>all-trans retinal</td></tr><tr><td>FuGENE HD</td><td>Promega</td><td>E2312</td><td>Transfection reagent</td></tr><tr><td>DMEM</td><td>Biochrome</td><td>FG 0445</td><td>Dulbecco&#039;s Modified Eagle Medium</td></tr><tr><td>Agarose</td><td>Roth</td><td>3810</td><td>Agar bridges</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>5239</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>CsCl</td><td>Biomol</td><td>2452</td><td></td></tr><tr><td>EGTA</td><td>Roth</td><td>3054</td><td></td></tr><tr><td>FBS</td><td>Biochrome</td><td>S0615</td><td>Cell culture</td></tr><tr><td>Glucose</td><td>Roth</td><td>HN06</td><td>D(+)-Glucose</td></tr><tr><td>KCl</td><td>Roth</td><td>6781</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>2189</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>NaCl</td><td>Roth</td><td>3957</td><td></td></tr><tr><td>NMG</td><td>Sigma Aldrich</td><td>M2004</td><td>N-Methyl-D-glucamine</td></tr><tr><td>Na-Aspartate</td><td>Sigma Aldrich</td><td>A6683</td><td>L-Aspartic acid sodium salt monohydrate</td></tr><tr><td>Citric acid</td><td>Roth</td><td>6490</td><td></td></tr><tr><td>AgeI</td><td>ThermoFischerScientific</td><td>ER1462</td><td>Restriction enzyme</td></tr><tr><td>XhoI</td><td>ThermoFischerScientific</td><td>ER0695</td><td>Restriction enzyme</td></tr><tr><td>NheI</td><td>ThermoFischerScientific</td><td>ER0975</td><td>Restriction enzyme</td></tr><tr><td>XL1Blue &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies</td><td>200249</td><td>Chemocompetent &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Kanamycin</td><td>Roth</td><td>T832</td><td></td></tr><tr><td>Lysogeny broth medium</td><td>Roth</td><td>X964</td><td></td></tr><tr><td>Agar-Agar</td><td>Roth</td><td>6494</td><td>Agar plates</td></tr><tr><td>Plasmid purification kit</td><td>Marchery-Nagel</td><td>740727.25</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Biochrome</td><td>A 2213</td><td>Cell culture</td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td>Cover slip coating</td></tr><tr><td>Microforge</td><td>Custom made</td><td></td><td>Fire polishing</td></tr><tr><td>Serological pipettes</td><td>TPP</td><td></td><td>Different sizes</td></tr><tr><td>Clean bench</td><td>Kojair</td><td>Biowizard SL130</td><td></td></tr><tr><td>Stirrer</td><td>IKA</td><td>RCT classic</td><td></td></tr><tr><td>Silver wire</td><td>Science Products</td><td>AG-T25; AG-T10</td><td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td></tr><tr><td>pH-meter</td><td>Knick</td><td>765 Calimetric</td><td></td></tr><tr><td>Osmometer</td><td>Vogel</td><td>OM 815</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>ID03</td><td>Fire polishing</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Binder</td><td>CB150</td><td></td></tr><tr><td>Cell culture dishes</td><td>TPP</td><td>93040</td><td>34 mm internal diameter</td></tr><tr><td>Cover slips</td><td>Roth</td><td>P232</td><td>15 mm diameter</td></tr><tr><td>Thermometer</td><td>R&amp;ouml;ssel Messtechnik</td><td>MTM12</td><td></td></tr><tr><td>Beamsplitter</td><td>Chroma</td><td>21011</td><td>90/10 transmission</td></tr><tr><td>Pipette holder</td><td>ALA Scientific Instruments</td><td>PPH-1P-AXU-0-1.5</td><td></td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>AxoPatch200B</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>DigiData1400</td><td>Digital analog converter</td></tr><tr><td>Lightsource</td><td>TILL Photonics</td><td>Polychrome V</td><td>Set to 540 nm full intensity</td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>Axiovert 100</td><td></td></tr><tr><td>Shutter</td><td>Vincent Associates</td><td>VS25</td><td></td></tr><tr><td>Shutter driver</td><td>Vincent Associates</td><td>VCM-D1</td><td></td></tr><tr><td>Glass capilarries</td><td>Warner Instruments</td><td>G150F-3</td><td>Boresilicate capillaries with fire polished ends OD 1.5 mm ID&amp;nbsp; 0.86 mm</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P1000</td><td></td></tr><tr><td>Bath handler</td><td>Lorenz Messger&amp;auml;tebau</td><td>MPCU</td><td></td></tr><tr><td>Tripleband filterset</td><td>Chroma</td><td>69008</td><td>Fluorescence filter&amp;nbsp; ECFP/EYFP/mCherry&amp;nbsp;</td></tr><tr><td>CCD camera</td><td>Watec</td><td>Wat-221SCCD</td><td></td></tr><tr><td>Optometer</td><td>Gigahertz Optik</td><td>P9710</td><td>Measure light intensities</td></tr><tr><td>Objective</td><td>Carl Zeiss</td><td>421462-9900-000</td><td>W Plan-Apochromat 40X/1.0 DIC</td></tr><tr><td>Micromanipulator</td><td>Scientifica</td><td>PatchStar</td><td></td></tr><tr><td>Recording chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power supply</td><td>Manson</td><td>HCS-3202</td><td>Avoids electrical noise from microscope built-in power supply</td></tr><tr><td>Vibration isolated table</td><td>Newport</td><td>M-VW-3636-OPT-01</td><td></td></tr><tr><td>Faraday cage</td><td>Custom made or any commercial matching table</td><td></td><td></td></tr><tr><td>Hoses</td><td>Any comercial; e.g. Roth</td><td></td><td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td></tr><tr><td>Linear shaker</td><td>Sunlab Instruments</td><td>SU 1000</td><td></td></tr><tr><td>Liquid junction potential calculator</td><td>Molecular Devices or directly from Peter H. Barry</td><td></td><td>Program is included in the Clampex aquisition software or can be obtained from&amp;nbsp; p.barry@unsw.edu.au</td></tr><tr><td>Data acquisition software</td><td>Molecular Devices</td><td>Clampex 10.X</td><td></td></tr><tr><td>Data evaluation software</td><td>Molecular Devices</td><td>Clampfit 10.X</td><td></td></tr><tr><td>&lt;em&gt;Ps&lt;/em&gt;ACR1</td><td>GenBank or Addgene</td><td>KF992074.1 or Addgene plasmid #85465</td><td>Gene encoding for &lt;em&gt;Ps&lt;/em&gt;ACR1</td></tr><tr><td>Amplifier guide</td><td>Molecular Devices</td><td>The Axon Guide</td><td></td></tr></tbody></table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

지난 10 년 동안 channelrhodopsins은 신경 세포 학적 연구에서 없어서는 안되게되었고, 대상 세포에서 전기 과정을 비 침습적으로 조작하는 도구로 사용되었습니다. 이러한 맥락에서, 채널로도 딘의 이온 선택성은 특히 중요하다. 이 기사는 최근 HEK293 세포의 전기 생체 학적 패치 클램프 기록을 통해 Proteomonas sulcata 의 최근 확인 된 음이온 전도성 채널로도 딘에 대한 염화물 선택성에 대한 조사를 기술 합니다. 빛에 의해 제어되는 광전류를 측정하기위한 실험 절차는 기존의 패치 - 클램프 설정의 현미경에 결합 된 신속하게 전환 가능한 이상적인 단색 광원을 필요로합니다. 실험 이전의 준비 절차는 완충 용액의 준비, 액체 접합 전위에 대한 고려, 세포의 파종 및 형질 감염 및 패치 피펫의 당김과 관련하여 개략적으로 설명됩니다. 전류 - 전압 관계의 실제 기록transfection 후 24 시간에서 48 시간 사이에 다양한 염화물 농도에 대한 reversal potential을 결정합니다. 마지막으로, 전기 생리 학적 데이터는 염화물 전도의 이론적 고려 사항과 관련하여 분석된다.

<html> 그림 2 는 설명 된 프로토콜에 따라 측정 한 결과를 보여줍니다. 초록색 조명으로 조명하는 동안 Ps ACR1은 빠르게 일시적인 전류 레벨로 빠르게 감쇄하는 빠른 과도 전류를 특징으로합니다. 빛이 꺼지면 광전류는 밀리 세컨드 내에 0으로 감소합니다 ( 그림 2A ). 세포 외 염화물 농도를 바꾸면 획득 한 광전자 흔적에서 직접 볼 수있는 역전 전위가 이동합니다. 다중 측정으로부터의 역전 전위의 평가는 외부 염소 농도의 변화에 ​​의해 야기 된 극적인 역 전위 변화를 정량화합니다 ( 그림 2B ). 놀랍게도, 측정 된 반전 전위는 클로라이드의 계산 된 Nernst 전위와 직접적으로 일치한다 ( 그림 2C ). 이는 Ps ACR1의 높은 염화물 선택도를 확인한다. t &quot;fo : keep-together.within-page =&quot;1 &quot;&gt; <img alt="그림 2" src="/files/ftp_upload/55497/55497fig2.jpg" /> 그림 2 : Ps ACR1 의 염화물 선택 도 . ( A ) HEK293 세포에서의 Ps ACR1의 대표적인 전류 추적. 세포 내 염화물 농도는 120 mM로 유지되는 반면, 세포 외 농도는 NaCl과 Na- 아스파 테이트의 부분 치환으로 150 mM (녹색)에서 10 mM (자주색) 염화물로 다양하다. 유지력은 -80 mV에서 +40 mV까지 20 mV 간격으로 증가했습니다 (LJP 보정 됨). ( B ) 평균 전류 - 전압 관계 (n = 6은 A의 조건에 해당). ( C ) 전류 - 전압 관계로부터 추출 된 역전 전위 (중심선은 중앙값, 박스 한계는 25 번째 및 75 번째 백분위 수, 휘발성 물질은 표준 편차의 2 배, 원은 단일 데이터 점을 나타냄). 염화 고밀도의 경우(녹색) 값은 -20 mV와 0 mV 유지 전위 사이에서 선형 적으로 보간되었지만 값은 +40 mV (보라색, 파선, 패널 B)에서 선형 적으로 외삽되었다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55497/55497fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Channelrhodopsins에서 이온 선택성의 전기 생리 학적 결정을위한 전체 셀 패치 - 클램프 기록

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

whole-cell-patch-clamp-recordings-for-electrophysiological

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

17.0K 조회수.  Humboldt-Universität zu Berlin. This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

비디오: Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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 ...

연구

생물학

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

양서류 망막 슬라이스 준비 광수와 2 차 신경 세포에서 동시에 전체 세포 녹음

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

신경과학

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

표현 이온 채널 연구에 대한 특정 감각 신경 세포 인구의 식별

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

표현과 HEK - 293T 세포의 재조합 전압 - 게이 티드 이온 채널 Electrophysiological 녹음에 최적화된 Transfection 전략

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법<em&gt; Drosophila</em&gt; 광 수용체

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

1 채널의 세포 부착 된 패치 클램프 녹음

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Source: Grimm, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55497">Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins.</a> J. Vis. Exp. (2017)
In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

1. Setup Prior to Recordings

	Prepare the recording solutions with ion concentrations as indicated in Table 1.&#8203;
	
		Prepare 15 mL of 2 M stock ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptor(초파리 광수용체의 전체 세포 전압 클램프 기록)

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

Electrophysiology Techniques

Patch Clamp

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively and negatively charged ions across the cell membrane. One competent method to record current flowing across the whole cell or single ion channel is the patch-clamp technique.
In this method, a glass micropipette containing electrolyte solution is tightly sealed against a small portion of the cell membrane. As a result, a patch of the cell membrane is electrically isolated, ensuring that the ions moving across the channels flow only into the micropipette. Further, this tight sealing of the cell membrane prevents ions from escaping into the bath solution in which the cell is suspended.
Different Patch-clamp Methods:
Depending upon the research interest, several variations of the patch-clamp method can be used for measuring the cell&#39;s biophysical properties. For example, researchers can clamp or control the membrane voltage and measure the current passing through it. Alternatively, they can clamp the current and measure any variation in the membrane voltage.
In cell-attached mode, the membrane patch containing a single or a few ion channels remains intact. As a result, the current flowing through the membrane patch alone can be measured. In contrast, the whole-cell method involves disruption of the membrane patch by briefly applying strong suction. Consequently, the interior of the pipette becomes continuous with the cytoplasm. This mode enables the measurement of electrical current and voltage from the entire cell.
Another patch-clamp method requires gentle retraction of the attached pipette. As a result, the membrane patch is excised without affecting the tight seal. In this inside-out configuration, the intracellular portion of the membrane is exposed to the bath solution, allowing the study of intracellular factors affecting the channel functions.

패치 클램프

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively ...