이 작품은 NIH 보조금 EY019312과 EY021126 (VJK), 워싱턴 대학 안과학 교실 및 Visual 과학에 EY002687에 의해 지원되었다, 연구에 의해 실명을 방지 할 수 있습니다.

모든 실험 프로토콜은 실험 동물의 관리 및 사용에 대한 가이드에 따라했고, 워싱턴 대학의 기관 동물 연구위원회에 의해 승인되었다. 1. 관류 및 시편 홀더 설정 <ol><li> 실험의 날에 신선한 망막 관류에 대한 솔루션을 준비합니다. 증류수 및 탈 이온수를 사용합니다. 다음 세 가지 방법 중 하나를 사용합니다. <ol><li> 미디어와의 NaHCO3 1.9 G : &#39;에임스 1 병 (1ℓ) 솔루션&#39;을 에임스를 중탄산염이 함유 준비 </li><li> (MM)에 로크의 솔루션 준비 : 112.5 염화나트륨, 3.6의 KCl, 2.4의 MgCl 2, 1.2 CaCl2를, 10.0 HEPES를, 20.0 NaHCO3를, 3.0 나 숙시 네이트, 0.5 나 조미료, 0.02 EDTA, 10.0 포도당, 0.1 % MEM 비타민과 아미노산 산 </li><li> (MM)에 준비 HEPES 완충 벨소리 솔루션 : 133.3 염화나트륨, 3.3의 KCl, 2.0의 MgCl 2, 1.0 CaCl2를, 10.0 포도당, 0.01 EDTA, 12.0 HEPES, pH를1 M의 NaOH 5.8 ml의 ~ 7.5로 조정, 0.72 G / L Leibovitz 문화 매체 L-15을 추가합니다. 어떤 관류 매체를 사용하여 광 수용체 응답을 분리하는 100 μm의 BaCl 2 - 50 μm의 DL-AP4 50 - 20를 사용합니다. </li></ol></li><li> (MM)에 전극 (28)에 대한 솔루션을 준비 : 140.0 염화나트륨, 3.6의 KCl, CaCl2를, 3 HEPES, 0.01 EDTA 2.4의 MgCl 2, 1.2 및 7.4에 pH를 조정 - NaOH로 7.5. 전극 액은 수개월 동안 실온에서 저장 될 수있다. </li><li> 준비하고 시편 홀더를 테스트합니다. <ol><li> 시험편 홀더의 바닥 부의 돔 위에 블랙 / 회색 여과지를 붙인다 (도 1A 참조). 돔 플랫 탑의 가장자리 주위 신중 이성 분 5 분 에폭시 접착제를 확산. 필요한 경우, 해부 현미경으로 그것을 할. </li><li> 접착제가 거의 건조 (약 4 분)이 될 때까지 기다렸다가 평면 항목을 사용하여 돔에 여과지를 누릅니다. 접착제 종이 필터 실험 전에 적어도 하루.여과지를 여러 번 사용할 수 있지만, 레코딩 개월 후에 교체되어야한다. 대체 용지를 설치하기 전에 접착제 잔류 물에서 조심스럽게 지붕을 청소하는 70 % 에탄올을 사용합니다. </li><li> 전극 액은 공기 방울을 방지하고 전극 채널로 나사산 어댑터로 둘러싸인 펠렛 전극 (도 1A 및 B 참조)하려고 나사가 구비 된 전극 채널을 채운다. 네 개의 나사와 시편 홀더의 상단과 하단 부분을 연결하고 전극 용액으로 관류 라인을 입력합니다. </li><li> 멀티 미터 (그림 1B)를 사용하여 각 전극 쌍의 리드 사이의 저항과 전압을 측정합니다. 저항 100 Ωk 10 MV 아래의 전압 채널 거품 무료이며 전극이 좋은 상태에있는 경우 이하이어야한다. </li></ol></li><li> 유리 병에 관류 매체의 700 mL의 - 400 붓는다. 또 다른 300 ㎖의 망막 및 S 해부에 사용되는 별도냉장고에서 그것을 찢었다. 열교환 블록 관류 튜브를 설정하고 가열판에 예열 된 블록을 배치 (도 1d 참조). </li><li> (그림 1D 참조) (에임스 &#39;또는 로크를 사용하는 경우) 연결이 2 / O 2 가스를 공동가 캡 병 및 관류 튜빙을 묶습니다. 37 O C의 미디어와 병을 예열 및 가열 플레이트 또는 37으로 설정 물을 욕조에 빛 자극 장치의 상단에 배치 - 39 O를 C (17). <ol><li> 국무 중력 중심의 흐름을 시작 관류 미디어를 작성하여 관류 라인. <ol><li> LKC 시스템에서, 다음 실험 기간 동안 병에 관류 용액의 저하 수준에 의해 영향을받지 않고, 유량 조절기에 의해 조절되는 중요한 중력 유동을 제공하도록 자극 부 (17)의 위쪽에 용기를 배치했다. </li><li> Ocuscience 시스템에서, P를 배치시편 홀더 긴 관류 출력 라인 노이즈를 최소화하고 배치하기 위해 패러데이 케이지 안에 erfusion 병 용액의 중력 구동을 증가시키는 잘 시험편 홀더 (관류 병)의 레벨 이하 (단계 2.8 참조). 이러한 출력 라인을 방패 ERG 신호에 전자 노이즈의 커플 링을 방지하도록 상기 증폭기의 접지에 실드를 연결한다. </li></ol></li></ol></li><li> 유량 조절기를 사용하여 / 분을 5 ml - 3 관류를 조정합니다. 적절한 규제와 실린더에서 5 % CO 2 / 95 % O 2 가스를 연결하고 공기 돌을 통해 병에 미디어의 꾸준한 버블을 보장하기 위해 유량을 조절합니다. </li></ol> 2. 샘플 준비 <ol><li> 직선 블레이 딩 microscissors, 하나 또는 두 개의 45 O 핀셋, 면도날 및 필터 종이의 사각형 조각을 포함하여 깨끗하고 날카로운 해부 악기를 조립합니다. </li><li> 감기 perfu의 약 200 ml에 붓고큰 페트리 접시에 시온 용액 (돔 포함) 시험편 홀더의 전체 바닥 부분 용액에 침지 될 수 있도록. 돔의 망막을 장착 할 때이 단계가 중요하게된다 (단계 2.6 참조). 일부 솔루션 carbogen (5 % CO 95분의 2 %의 O 2)로 포화되도록 설계되어 있지만, 이는 해부 상업적 필수적이지 여기 기재된 실험을 수행하지 않았다. </li><li> 녹음하기 전에 12 시간 - 전형적인 실험, 어두운 / 라이트 사이클과 6을 어두운 - 적응 12/12 시간에 동물을 유지. (현미경 광원 앞에 예를 들어, 빨간색 필터를 사용) 희미한 붉은 빛 아래 자궁 전위 다음 이산화탄소 흡입 동물을 안락사 및 어두운 빨간색 또는 적외선 조명 아래 모든 다음 절차를 수행합니다. <ol><li> 핀셋을 사용하여 눈을 잡아 당겨 미디어에 넣어. 페이퍼 (예를 들어, 일부 정규 여과지)의 작은 조각에 한번에 하나의 눈으로 만들어 놓고핀셋 눈 (이 여기에 솔루션의 외부에서 수행되는) 잡고 오라 세라의 수준에 약 조명. </li></ol></li><li> 오라 세라 따라 절단 (또는 가까운 눈의 적도에) microscissors와 각막과 렌즈를 제거합니다. 큰 페트리 접시에 차가운 미디어에서 아이 컵을 놓고 다른 눈으로 동일한 절차를 반복합니다. </li><li> 가능한 손상으로 망막을 유지하기 위해 망막과 공막 사이에 위를 유지하여 시신경 향해 아이 컵의 상단에서 작은 절개를 잘라. 그립 개의 핀셋을 사용하여 망막 분리를 위해 서로 떨어져 핀셋 당겨 의해 절개 양측으로부터 공막. <ol><li> 시신경을 잘라 말단 표면에 물리적 인 접촉의 최소 금액으로 망막을 분리하려고합니다. RPE 주로 박리 과정에서 자동 망막 분리한다. 그것은을 수행하는 것보다 망막의 기계적 교란을 피하기 위해 더 중요해부 빠르게 망막은 일반적으로 응답 특성에 심각한 영향없이 용액 중에서 적어도 30 분 동안 항온 처리 할 수​​있다. </li></ol></li><li> 시편 홀더 (그림 1C)의 돔에 망막를 탑재합니다. 해부 망막으로 페트리 접시에 시료 홀더의 바닥 부 담가. 돔 상기 망막 위쪽 감광체 측 (절연 망막의 볼록면)을 밀어 망막 여과지에 부착되도록 시험편 홀더를 해제. 다른 망막에 대해이 절차를 반복합니다. </li><li> 전기 망막 사이의 크로스 토크뿐만 아니라 노이즈와 신호 입환을 방지하기 위해주의 깊게 홀더 플레이트를 건조. 시험편 홀더는 홀더의 하부 및 상부 부분 사이의 액 유출을 방지 할뿐만 아니라 개별 망막의 감광체 및 신경절 세포 양쪽의 전기적 분리를 돕기 위해 바닥 부 돔 주위에 O 링을 갖는다. (네 개의 나사와 홀더의 상단 부분을 연결도 1b 참조), 주사기와 바늘을 이용하여 관류 용액으로 관류 채널을 채운다. </li><li> 열교환 블록 옆 시험편 홀더를 전송하고 시험편 홀더 (도 1D)에 대한 입력 및 출력 관류 라인을 연결한다. (상부 전극 증폭기 접지 / 마이너스 핀에 연결) ERG 증폭기에 전극을 연결하는 어댑터를 사용하여 표본 홀더에 자극기 / 제어 유닛을 부착하거나 따라 자극 부에 가열 패드를 밀어 생체 내에서 그 위에 에르그 시스템이 사용됩니다. </li></ol> 3. 녹음 <ol><li> 생체 시스템의 소프트웨어를 사용하여 앰프와 자극 설정을 구성합니다. 300 Hz로 필터링 1 ~ 10 kHz와 로우 패스 사이의 값으로 인수 주파수를 설정합니다. 하이 패스 필터링을 사용하지 마십시오. 60 Hz에서 (또는 유럽에서 50 Hz에서) 필요한 경우 필터를 홈을 사용합니다. </li><li> 빛 자극과 재치없이 기록 기준시간 희미한 자극 (예., -35 dB의 0.3 MCD의 SM -2의 녹색 빛) 전극과 망막 샘플 사이 좋은 전기적 연결을 테스트합니다. 망막 온도 함수 안정된 상태에 도달하도록 데이터 수집을 시작하기 전에 20 분 - 10 기다린. <ol><li> 특정 실험에 따라 자극 파라미터를 선택하고 데이터 수집을 시작합니다. 예를 들면, 0dB까지 0.1 1,000까지 MCD의 SM에서 -40에서 녹색 빛을 사용 -2 암순응 응답 가족을 위해. 봉 배경 빛에 의해 억제 할 필요가 포토 픽 녹음에 약 2 밝은 로그 단위 3 깜박 사용 (3-30 CDM -2) 또는 프로브 플래시 (-2 = 1dB 약 1.3 카드뮴 SM을, 그림 3 참조). </li><li> 그러나 정확한 빛의 강도 값이 시스템 교정 및 실험 조건에 다소 의존 할 수 있음을 기억하십시오. 경험적으로,도 5에 의해 다운 스케일 강도 - 10 배는 생체에 비해 </eM&gt; 녹음 녹색 빛 (17)를 사용하여. (상업적 어댑터 시스템에 포함) 망막 위의 개구를 가진 블랙 커버 시험편 홀더에 광 산란을 감소시키고 시스템이 생체 내에서 상용의 Ganzfeld 구함으로써 두 망막의 균일하고 동일한 자극을 용이하게하는데 사용될 수있다. 참고 : 어두운 적응 망막에서 녹음 들어, 일반적인 실험 표백제 안료의 무시할 부분에 사용되는 테스트 깜박의 강도가되도록 망막 색소 상피 중심의 재생의 부족은 문제가되지 않습니다. 또한, 이전에 콘 안료 재생 여전히 뮐러 세포주기 (20)를 통하여 시각적 절연 망막에서 일어날 수 있음을 보여왔다. </li></ol></li><li> 실험은 몇 시간에 30 분부터 일반적으로 마지막으로 이러한 변화는 기술적 인 문제를 나타낼 수로, 실험 기간 동안베이스 라인 드리프트, 소음 및 응답의 안정성을 모니터링 할 수 있습니다. 참고 : 노이즈 증가 또는 감소D 응답 진폭은 공기 관류 또는 전극 채널 거품, 너무 낮거나 높은 온도, 용액 누출 / 유출 또는 망막의 변위를 표시 할 수있다. </li></ol> 4. 청소 <ol><li> , 관류 라인으로부터 시험편 홀더를 분리하여 열고 여과지로부터 망막 플러시. 전극을 제거하고 증류수 (에탄올을 사용하지 않는)로 씻어. 에탄올 및 / 또는 증류수 (관류 채널 포함) 시편 홀더를 청소합니다. 조심스럽게 세척 관류 관 70&gt;와 % 에탄올. </li><li> 증류수 (세제를 사용하지 마십시오)와 관류 병을 씻어. 에탄올과 같은 병을 세척하기 위해 사용될 수있다. </li></ol>

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세인트 루이스의 워싱턴 대학은 Xenotec, 사와 라이센스 계약을 가지고 있으며, 생체 어댑터의 판매 로열티를받을 수 있습니다.

<html> 우리는 여기서 생체 ERG 어댑터와 함께 생체 ERG 시스템 구성 요소를 사용하여 두 개의 격리 된 마우스의 망막 동시에 고품질 생체 ERG 녹화를 얻기위한 중요한 단계를 보여준다. 본 연구에서 우리는 동일한 용액 (어느 에임스 &#39;, 로크 또는 링거)과 동물 모두에서 망막 관류하지만 약물 테스트를 위해 상이한 솔루션 등 각 망막 관류하는 것도 가능하다. 고품질...

전위도 (ERG)은 빛에 의해 트리거 망막의 전기 활동을 기록 할 수있는 잘 확립 된 기법이다. ERG 신호는 망막의 세포 외 공간 저항에 흐르는 전류에 의한 방사 전압 변화 (감광체 바이폴라 셀의 축 방향)에 의해 주로 생성된다. 제 ERG 신호 은점 (1)의 표면으로부터 그렌에 의해 1865 년에 기록되었다. Einthoven 유쾌한 1908 2 B-, A-라는 세 가지 파도에 빛의 발병 ERG 응답, 및 C 파 지금 쌍극 세포, 감광체 주로 활동을 반영하는 것으로 알려져 있으며, 색소 상피를 분리 세포, 각각 3-8. 에르그 지역 (미소 전극과 (생체) 고립 그대로 망막에 걸쳐, 고립 된 눈 준비 (9)에서, 마취 동물 또는 (생체 내) 인간의 눈에서 3,10-15 또는에서 특정 망막 층을 기록 할 수 있습니다ERG) 4,16. 이들 중에서, 생체 ERG 현재 망막 기능을 평가하기 위해 가장 널리 사용되는 방법이다. 또한 진단 목적으로 이용 될 수 있거나 또는 동물이나 환자의 망막 질환의 진행을 따라 비 침습적 방법이다. 그러나 생체 내 에르그 녹음은 종종 안구 생리 소음 (예, 호흡과 심장 활동)에 의해 오염, 여러 중복 구성 요소와 복잡한 신호를 생성한다. 로컬 ERG는 망막의 특정 층에 걸쳐 신호를 기록하기 위해 사용될 수 있지만, 가장 침습적이고 다른 ERG 기록 구성과 비교하여 낮은 신호대 잡음비 (SNR)를 갖는다. 지역 망막 전위도 기술적으로 요구하고 고가의 장비 (예를 들어, 현미경과 미세 조작기)가 필요합니다. 그대로 고립 된 망막에서 Transretinal ERG (생체 ERG)는 안정적이고 HIG을 허용 생체 내 및 지역 에르그 방법 사이의 타협을 제공합니다동물이나 인간 (17)의 손상 망막에서 시간 SNR 녹음. 최근에,이 방법은 포유 동물, 영장류 및 인간 망막 18-20에 막대와 콘 감광체 기능을 연구하기 위해 성공적으로 사용되어왔다. 또한, 인해 생체 망막 색소 상피 세포의 부재로, ERG 신호의 포지티브 형 C 파 성분을 제거하고 눈에 띄는 네거티브 느린 PIII 성분은 생체 녹화 드러난다. 느린 PIII 성분은 망막 21-23에서 뮐러 아교 세포의 활성에서 발생하는 것으로 나타났다. 따라서, 생체 외 ERG 방법도 그대로 망막 뮐러 세포를 연구하기 위해 사용될 수있다. 여러 연구는 또한 생체 ERG 레코딩은 망막 (24) 주위되는 약물의 농도를 측정하고, 약 25 ~ 27의 안전성과 효능을 테스트하는 데 사용될 수 보여 주었다. 생체 시스템의 여러 상업이 가능하며,반드시 광범위한 전기 생리학 배경이없는 많은 실험실에서 사용. 대조적으로, 체외 장치 (17)는 최근까지 극소수 실험실은이 강력한 기술을 활용되는 결과로서 사용할 수 없었다. 그것은 망막의 생리와 병리에 대한 우리의 지식을 향상하고, 질병을 눈부신에 대한 새로운 치료법을 개발하기 위해 더 많은 실험실에 생체 에르그 녹음을 사용할 수 있도록하는 것이 도움이 될 것입니다. 우리는 여기에 간단하고 저렴한 생체 ERG 장치 (17)을 설명하고는 일단이 상기 조작 패널 및 원뿔 - 매개 신호를 기록하는 몇몇 시판 생체 ERG 시스템과 조합하여 사용될 수있는 방법을 보여준다 (A- 및 B- 파)와 함수 그대로 야생형 마우스 망막에서 뮐러 세포 (PIII을 느리게).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

우리는 암순응 야생형에서 플래시 응답을 기록 실험 프로토콜 표준 관류 다른 해결책 (도 2)를 사용하여 상술 한도 1에 도시를 수행하여 (WT) C57BL / 6 마우스의 망막. 응답 파형과 반응 속도뿐만 아니라로드 광 수용체의 민감도는 에임스 &#39;와 로크의 미디어 (그림 2A와 B)에서 유사 나타났다. 한편, HEPES 완충 링거액 (NO 중탄산염 또는 5 % CO 95분의 2 %의 O <...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.0K 조회수.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

비디오: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

연구

신경과학

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Polyribosome 분별에 의한 중추 신경계 Translatome의 생체 심문

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

본래 Vomeronasal 기관 및 액세서리 후각 망울의 생체 준비

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

뇌 조각의 공포 회로의 예 생체 Optogenetic 해부

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 가압 해모골 모세관-Parenchymal Arteriole 기능 적 연구를 위한 준비

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

경동공 이광자 마우스 망막의 생체 내 이미징

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

작고 큰 눈의 망막 기능을 연구하기 위해 생체 외 망막 전도의 설정 및 조건 최적화

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

생체 외 설치류의 삼차 혈관 시스템에서 칼시토닌 유전자 관련 펩타이드의 방출

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...