This work was supported by the World Class Institute (WCI) program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology of Korea Grant WCI 2009-003 and Korea Institute of Science and Technology Institutional Program Project 2E24210. Sungmoo Lee was supported by Global Ph.D. Fellowship program (NRF-2013H1A2A1033344) of the National Research Foundation (NRF) under the Ministry of Education (MOE, Korea).

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저자는 공개 아무것도 없어.

신경 조직은 여러 가지 방법으로 전압이 약간 억제 과분극가 시냅스 입력이 비교적 큰 전압 변화에 약간의 탈분극 및 활동 전위 결과 발생 원인 이용한다. GEVIs하여 막 전위의 변화를 측정 할 수 동시에 신경 회로의 여러 구성 요소를 분석 유망한 잠재력을 제공한다. 이 보고서에서 우리는 GEVIs를 사용하여 막 전위의 이미지 변화를위한 기본적인 방법을 보여줍니다. 전압 이미징 변경 주요 키 세포막에서 GEVI의 효율적인 표현이다. 세포 내 발현은 프로브의 SNR을 감소시키는 비 형광 반응을 생성한다. 형질 전환 조건의 최적화는 크게 광 측정의 일관성을 개선한다. 소설 GEVI을 테스트 할 때 실제로, 광학 활성의 차이는 북동에 전적으로 기인 있는지 확인하는 것도 알려진 프로브를 테스트하는 것이 좋습니다w 프로브 셀없는 상태. 도 6은 전압 촬상의 감도를 감소 내부 형광의 효과를 나타낸다, ΔF / F 값.도 6b는 ΔF는 단일 FP 기반 GEVI, Bongwoori 표현 HEK 세포 값이다. 추적이 상대적으로 밝은 내부 형광도 6A에서 볼 수 영역 (2) (청색)에서 비롯됩니다. 이 추적 인해 ΔF / F를 감소 밝은 내부 형광에 2 방울 크게도 6b의 흔적이 ΔF / F 값에 대한 RLI 값으로 나누어 그림 6C, 추적에서, 그러나 추적 1과 ΔF 값과 비슷한 수준이 값. 형광 추적 (3)는 매우 작은 ΔF이뿐만 아니라 아주 작은 RLI 값 (F)가 있습니다. 그 결과, 기록 노이즈에 상당한 증가가있는 잘못된 ΔF / F 신호이다. 또한이 예제는 ΔF을 보여주기 위해 포함되었다중대한. F로 시작하는 것이 매우 낮은 경우 ΔF / F에서 큰 변화는 도움이되지 않습니다. 영상 분할의 사용은 하나의 CCD 카메라로 2 개의 파장의 측정을 수반 할 수있다. 이것은 크게 프로브 개발 FRET 종속 형광 변화의 측정을 지원하고, 설치 비용을 감소시킨다. 그러나, 색수차 때문에,이 두 이미지는 아포 크로매틱 목적에 대한 필요성을 필요로 약간 초점이 될 것입니다. 그래서 가장 높은 개구와 목표를 선택하는 더 많은 빛 더 나은 SNR은 또한 형광 이미징을 향상시킨다. 또한, 하나는 발광 다이오드 (LED) 또는 SNR을 증가시키는 레이저로 강한 광원을 사용할 수있다. LED는 기계적 셔터를 필요로하지 않습니다. 이 보고서에서 우리는 하나의 FP GEVI과 FRET 기반 GEVI에서 광 신호를 보여줍니다. FRET 기반 GEVI의 가장 큰 장점은 비율 적 영상은 상관 잡음 D의 감소를 수 있다는 것입니다생체 내 호흡과 혈액 흐름에 UE. 형광 신호의 반대 극성 막 전위의 진정한 변화를 확인한다. 두 발색단의 형광 강도가 종종 상당히 다를 때문에, SNR은 달라질 수 있습니다. 이 비율 계량 분석을 교란하고 그 유용성 (28)을 제한한다. 또한 FRET 독립적 인 신호 (29)가있을 수 있습니다. 따라서 때로는 더 나은 막 전위의 변화를 분석하기 위해 FRET를 사용하는 경우에만 밝은 형광 발색단을 사용하는 것입니다. 전압 이미징 때문에 속도 및 칼슘 플럭스를 측정 칼슘 이미징에 비해 막 전위의 변화 변이 칼슘 이미징보다 더 도전이다. 뉴런 막을 칼슘 이온 플럭스에 비해 매우 빠른 시간 척도에서의 전위 변화, 및 서브 임계 이벤트 칼슘 이미징 30으로 측정 될 수 없다. 또한, 전압 프로브는 세포막에 있어야막 전위에서 광학적 리포트 변경 가능한 프로브의 양을 감소시키는 효과가있다. 따라서, 실제로는 CCD 칩에 도달 할 광자의 수는 상대적으로 소수이다. 이것은 높은 양자 효율 및 막 전위의 변화에​​ 따라 높은 SNR을 이끌어 프로브 고속 낮은 판독 노이즈 카메라의 필요성을 요구한다. 시험 관내에서 세포를 묘화함으로써, 연구자들은 더 생체 측정에서 전에 GEVIs의 전위 장단점을 이해할 것이다.

본 연구의 주요 초점은 유전자 코드 형광 단백질을 이용하여 시험관 내에서 막 전위 변화 결상을 입증하는 것이다. 막 전위의 이미지 변화는 신경 회로의 활동을 연구의 흥미로운 가능성을 제공합니다. 형광 강도의 변화에​​ 따라서 막 전위 변화는 카메라의 각 화소는 비 침입 신경 활성의 측정을 가능하게되면 서로 게이트 전극. 40 년의 경우, 유기 전압에 민감한 염료는 1-4 잠재적 인 멤브레인의 변화를 관찰하는 데 유용되고있다. 그러나, 이들 염료는 세포 특이성이 부족하다. 또한, 일부 세포 유형은 염색하기 어렵다. 유전자 인코딩 된 전압 지표 (GEVIs)는 세포가 구체적으로 연구 형광 전압에 민감한 프로브를 표현하는 데 이러한 한계를 극복. GEVIs의 세 가지 클래스가 있습니다. GEVI의 첫 번째 클래스는 VO를 사용단일 형광 단백질 (FP) 5-9이나 포스터 공명 에너지 전달 (FRET) 쌍 10-12 중 하나와 전압 센싱 포스파타제 도메인에서 전 압을 감지. 센서의 두 번째 클래스는 형광 표시 직접적으로 13-15로 또는 전기 FRET (16, 17)를 통해 미생물 로돕신을 사용합니다. 세 번째 클래스는 두 개의 구성 요소, 막 고정 된 FP 및 막 결합 담금질 염료 18 ~ 20 인 두 번째 구성 요소 인 유전 적 구성 요소를 사용합니다. 두 번째와 세 번째 클래스가 시험 관내 및 슬라이스 실험 (19, 20)에 대한 유용하지만, 센서의 첫 번째 클래스는 현재 생체 내 6를 분석하는 데 유용합니다. 이 보고서에서 우리는 시험 관내에서 GEVIs의 첫 번째 클래스 (그림 1)를 사용하여 막 전위의 영상을 보여줄 것입니다. 전압 센서의 첫 번째 클래스는 생체 내 이미징로 전환 할 수있는 가장 쉬운 방법입니다. GEVIs U 이후약 센서 로돕신 클래스보다 밝게 50 배 FP 융합 전압 감지 영역이다 tilizing 그들이 아크 램프 조명을 이용하기보다는 매우 강력한 레이저를 필요로 묘화 될 수있다. 휘도 불균형의 또 다른 결과 GEVIs의 일류 쉽게 뇌의 자동 형광을 초과 할 수 있다는 것이다. 로돕신 기반 프로브는 할 수 없습니다. 센서의 세 번째 클래스는 첫 번째 클래스만큼 밝은하지만 생체 내에서 관리하기 어려운 화학 소광의 추가가 필요합니다. 그러므로 우리는 하나의 FP (Bongwoori) 8 FRET 쌍으로 구성된 프로브 (나비 2) (12)와 프로브의 인수를 시연 할 예정이다. 나비 2.242과 나비 2.244라는 이름의 녹색 형광 기증자, 클로버로 구성된 11, 빨간 형광 수용체, mRuby2 - VSFP-CR (클로버 - mRuby2 전압에 민감한 형광 단백질)의 나비 버전은 FRET이 보고서에 구축되어 <s&gt; 12입니다. 녹음 이러한 유형의 도입은 연구자에게 제공 할 정보의 유형 GEVIs의 더 나은 이해를 제공한다. <img alt="그림 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> 그림 1.이 보고서 (A)에 군데 유전자 인코딩 된 전압 표시 (GEVIs) 횡단 막 전압 감지 도메인과 형광 단백질을 갖는 GEVI 기반의 모노 FP의 두 가지 유형. (B) 횡단 막 전압 감지 도메인, FRET 공여체와 수용체로 구성 FRET 기반 GEVI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">IX71</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60X objective lens (numerical aperture = 1.35)</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">UPLSAPO 60XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">&#160;FF02-472/30</td>
			<td rowspan="3" style="width:187px;">For voltage imaging of super ecliptic pHluorin in Bongwoori</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-497/LP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">75W Xenon arc lamp</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">OptoSource Illuminator</td>
			<td style="width:187px;">LEDs and lasers are also effective light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Slow speed CCD camera</td>
			<td style="width:165px;">Hitachi</td>
			<td style="width:203px;">KP-D20BU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dual port camera adaptor</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">U-DPCAD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High speed CCD camera</td>
			<td style="width:165px;">RedShirtImaging, LLC</td>
			<td style="width:203px;">NeuroCCD-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image splitter</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">Optosplit 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-475/23-25</td>
			<td rowspan="5" style="width:187px;">For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET520/40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF560-FDi01-25X36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET645/75</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Vibration isolation system</td>
			<td style="width:165px;">Kinetic systems</td>
			<td style="width:203px;">250BM-IC, 5702E-3036-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patching chamber</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">RC-26G, 64-0235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">#0 Micro Coverglass (22x40mm)</td>
			<td style="width:165px;">Electron Microscopy Sciences</td>
			<td style="width:203px;">72198-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Temperature controller</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">#0 (0.08~0.13mm) - 10mm diameter glass coverslip</td>
			<td style="width:165px;">Ted Pella</td>
			<td style="width:203px;">260366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lipofection agent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">11668-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcium phosphate reagent</td>
			<td style="width:165px;">Clontech - Takara</td>
			<td style="width:203px;">631312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patch clamp amplifier</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">EPC 10 USB amplifier</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Multi-channel data acquisition software</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">Patchmaster</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image acquisition and analysis software</td>
			<td style="width:165px;">RedShirtImaging</td>
			<td style="width:203px;">Neuroplex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Spreadsheet application software</td>
			<td style="width:165px;">Microsoft</td>
			<td style="width:203px;">Microsoft Excel 2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Data analysis software</td>
			<td style="width:165px;">OriginLab</td>
			<td style="width:203px;">OriginPro 8.6.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Demagnifier</td>
			<td style="width:165px;">Qioptiq LINOS</td>
			<td style="width:203px;">Optem standard camera coupler 0.38x SC38 J clamp</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Confocal microscope</td>
			<td style="width:165px;">Nikon</td>
			<td style="width:203px;">Nikon A1R confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Anti-fade reagent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">P36930</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging membrane potential with two types of genetically encoded fluorescent voltage sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

일시적으로 형질 감염된 세포는 크게 형광 강도의 변화 및 ​​세포막 발현의 정도를 나타낼 수있다. 심지어 같은 coverslip에 일부 세포는 내부 형광의 다양한 수준을 제공합니다. 이는 세포에 의해 흡수 형질 제의 양 가장 쉽다. 때때로 너무 많이 발현이 세포 사멸 (27)의 결과로 펼쳐진 단백질 응답을 체험 할 수있는 세포 발생 (밝은, 높은 내부 형광, 세포를 반올림). 실험 따라서 내부 식 잡음비 (SNR)에 신호를 저하 무응답 형광을 생성 이후 가능한 한 적은 내부 형광 밝은 세포 희미 세포 모두를 테스트 할주의한다. 또 다른 고려 사항은 그림 3. 발색단의 성숙 속도 인 수용체 chromoph의 형광에서 차이를 보여줍니다공여 발색단 (클로버)보다 긴 숙성 시간이 광석 (​​mRuby2). <img alt="그림 3" src="/files/ftp_upload/53566/53566fig3.jpg" /> HEK 293 세포의 그림 3. 공 촛점 이미지는 일시적으로 FRET 기반 GEVI, 형질 Nabi2.242. (A) (B)의 잠재적 인 결과를 보여주는 공 촛점 이미지를 FRET 기증자 및 수용체에서 막 지역화 된 형광을 나타내는 HEK 세포의 공 촛점 이미지 mRuby2의 느린 성숙. 두 전시 mRuby2 형광이. 동안 모든 4 개의 셀은 클로버 형광을 나타내는 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 이미지는 공 초점 현미경에 의해 인수되었다. FRET 도너를 들면, 클로버, 488 nm의 레이저 여기에 사용 된 상기 발광은 CAPT시켰다ured와 50분의 525 nm의 대역 통과 필터. FRET 수용체, mRuby2가, 561 nm의 여기 레이저 및 50분의 595 nm의 대역 통과 필터를 조명 하였다 배출을 위해 사용되었다. 공 촛점 이미징을위한 샘플은 pH를 7.4로 조정 인산염 완충 식염수에 4 % 파라 포름 알데히드 / 자당 용액으로 고정하고 방지 페이드 시약 장착되었다. 형질 전환 조건이 최적화되면, 변이 다음 소스 분석하는 관심 영역을 결정하는 단계에서 온다. 높은 형광 변화의 셀 영역을 식별하기 위해 프레임을 사용하는 것은 감산 종종 ΔF / F 값을 최대화하기 위해 사용된다. 다른 셀로부터의 광을 수신하는 모든 화소를 선택한다. 이것은 잡음의 감소를 초래 화소 수를 증가 시키지만, 무응답 내부 형광이 포함되기 때문에, 신호의 크기를 감소시킨다. 두 방법 모두 한 실험자의 일관성 유지되는 정상입니다. <pFO 클래스 = &quot;jove_content&quot;킵 together.within 페이지 = &quot;1&quot;&gt;도 4a는 단일 FP 기반 GEVI, Bongwoori 표현 HEK 세포의 형광 변화를 나타낸다. 이 계단 전압 펄스에 응답하여 일반적인 형광 변화이다. 이 데이터에서 하나 GEVI의 전압 감도를 그릴 수에 단일 또는 이중 지수 감쇠 함수에 피팅에 의해 서로 다른 전압에서 τ 상수 오프를 결정합니다. 작은 전압 스텝의 SNR을 개선하기 위해, 기록 중에 잠재적 표백을 검출하는 HEK 세포를 이미지화 할 때 (16)는 전형적으로 시험 평균화된다. 100 MV에 강력한 신호를주고 그 프로브는 해리 해마 신경 세포 (그림 4C)에서 테스트 할 수 있습니다. 해마 신경 세포에서 형광 추적은 하나의 실험이다. <img alt="그림 4" src="/files/ftp_upload/53566/53566fig4.jpg" /> 단일 FP 기반 GEVI 그림 4. 전압 이미징 표현HEK 293 세포 및 주요 해마 뉴런. 고속 CCD 카메라와 1 kHz에서 기록 단차 전압 펄스에 대한 응답을 도시 한 FP 기반 GEVI, Bongwoori의 (A) ΔF / F 트레이스한다. (B), 고속의 CCD 카메라로 촬상 HEK 293 세포; Bongwoori 및 형광 변화를 관찰 하였다 화소를 나타낸다 (오른쪽)의 프레임 차분 화상을 표현하는 세포 (왼쪽) 휴식 광량 (RLI). (C) Bongwoori를 발현하는 마우스 해마 차 신경 세포에서 유도 된 활동 전위의 광 기록. 활동 전위는 전체 셀 전류 클램프 모드에서 유발 하였다. ΔF / F 추적이 소마 상관 관계 픽셀에서 선정되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 대표 FLUO도너 및 억 셉터의 발색단 rescence 판독은도 5a에 도시되어있다. 비율 적 분석을 가능 도너 및 억 셉터는 상관 잡음 소스, 예를 들면., 이동을 감소시키기 위해 형광 변화의 극성은 반대이다. 예를 들어, 이동 상관 잡음 모두 공여체 및 형광 수용체에 대해 동일한 방향으로 형광의 변화를 나타낼 것이다. FRET 프로브를 비율 적 기반 이미징 할 동안 만 밝은 발색단을 분석하는 것이 좋다. 디머 발색단 실질적 기록 노이즈를 증가시킬 수 있기 때문이다.도 5b는이 효과를 보여준다. 광 신호는 셀이고 밝은 클로버로부터도 5b에서 프레임 감산 분명히 보여준다. 반대로 mRuby2 형광 프레임 감산 셀에 걸쳐 높은 정도의 잡음을 나타낸다. 따라서 클로버의 도너 신호는 인터넷에서 해마의 신경 세포에 도시gure의 5C. <img alt="그림 5" src="/files/ftp_upload/53566/53566fig5.jpg" /> GEVI 기반의 FRET 그림 5. 전압 영상은 HEK 293 세포와 해마 차 신경 세포로 표현. (A) 고속의 CCD 카메라와 1 kHz에서 기록 단차 전압 펄스에 대한 2 개의 파장에서의 응답을 도시 FRET 기반 GEVI, Nabi2.242의 ΔF / F 추적. (B) 최고; 기증자 (클로버 - RLI, 클로버 프레임 공제)와 수용체 (mRuby2 - RLI, mRuby2 프레임 공제) 각 FP, 아래 두 개의 서로 다른 임계 값으로 처리 된 이미지; 같은 임계 값은 mRuby2의 상대 어스레을 설명하기 위해 두 기증자 (클로버 - RLI, 클로버 프레임 공제)와 수용체 (mRuby2 - RLI, mRuby2 프레임 공제) 이미지를 사용 하였다. 모든 이미지는 Nabi2.242을 표현 같은 HEK 293 세포에서 촬영되었다. (C) 520 nm의 파장 TRNabi2.244 센서를 발현하는 마우스 해마 차 신경 세포에서 유도 된 활동 전위의 에이스. ΔF / F 추적이 소마 상관 관계 픽셀에서 선정되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 6" src="/files/ftp_upload/53566/53566fig6.jpg" /> ΔF 및 ΔF / F 값에 대한 변하는 빛의 레벨 그림 6. 결과입니다. (A) 단일 FP 기반 GEVI, Bongwoori을 표현 HEK 293 세포의 이미지, 휴식 빛의 강도에 표시 (RLI), (B) 형광 추적 도시 커널은 평균 밝은 내부 형광을 가진 지역 : 잘 지역화 된 형광 신호, 지역 2 막 지역 : ΔF 세 가지 영역, 영역 1의 값 (F는 -F 0 X)D 영역 (3) :. 광 신호로부터 먼 지역, (C)는 커널을 보여주는 형광 흔적 (B)에서 같은 지역의 ΔF / F 값 평균 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 개념 </td><td> 식 </td><td> 말 </td></tr><tr><td> 분수 형광 변화 (ΔF / F) </td><td><img alt="식 (2)" src="/files/ftp_upload/53566/53566eq2.jpg" /></td><td> 시점에서 측정 F (1) = 빛의 강도는, F 0 = 빛의 강도는 유지 전위 측정 </td></tr><tr><td> 볼츠만 기능 </td><td><img alt="식 (3)" src="/files/ftp_upload/53566/53566eq3.jpg" /></td><td> Y = ΔF / F (MV)에서의 V는 = 막 전위 V 2 반 최대 ΔF / F, MV에서의 막 전위는 <sUB&gt; 1 = 최소값, 2 = 최대 값 DX = 기울기 </td></tr><tr><td> 두 지수 감쇠 기능 </td><td> Y = y를 0 + 1 전자 - (t - t 0) / τ 1 + 2 전자 - (t - t 0) / τ 2 </td><td> τ 1, τ = 2 시상수, 1, 2 = 진폭, t, t = 0 개의 시점은, Y = 0 오프셋 </td></tr></tbody></table> 표 2. 방정식

Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

유전자 인코딩 된 형광 전압 센서의 두 가지 유형의 잠재적 이미징 막

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

imaging-membrane-potential-with-two-types-genetically-encoded

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.6K 조회수.  Seoul National University. A method for imaging changes in membrane potential using genetically encoded voltage indicators is described. 

비디오: Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

전압에 민감한 염료와 크랩 Stomatogastric 신경절에서 뉴런의 광학 이미징

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

연구

신경과학

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

라이브 랫 두피 뉴런에서 Mitochondrial 분리막 잠재력과 반응 산소 종의 결정

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

영상의 연결 응답 Vomeronasal 조직의 슬라이스 준비에 유전적으로 인코딩된 칼슘 센서를 표현

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

동시 전압 클램프 및 Amperometry과 도파민 릴리스의 단일 세포 측정

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

막 성인 쥐에서 Ventromedial 시상 하부 뉴런의 잠재적 인 염료 이미징은 포도당 감지를 연​​구하는

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

이미징 세포 내 칼슘<sup&gt; 2 +</sup&gt; 유전자 인코딩 된 칼슘 표시기를 사용하여 성인 마우스의 선조체 성상의 신호

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

마우스 척수의 세포 역학의 두 광자 이미징

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

뉴런의 유전자 인코딩 된 부 자연스러운 아미노산을 사용하여 신경 세포 단백질의 광 제어

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.