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

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

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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

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

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

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			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
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			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
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			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
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			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
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			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
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			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
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			<td rowspan="2" style="width:88px;"></td>
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			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
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			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
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			<td style="width:88px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
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		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
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methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

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

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

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

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

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

마우스 부신하는 chromaffin 세포에서 세포 부착 된 정전 용량 측정을위한 방법

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

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

Research

JoVE Journal

Neuroscience

8.7K 조회수.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

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

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

마우스 Forebrain에 초음파 유도 Microinjection Utero에서 E9.5에서

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

연구

신경과학

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

여러 마우스 Neuroanatomical 자기 공명 영상

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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

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

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

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

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

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

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

마우스 모델에서 정맥 주사 자기 관리를위한 방법

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

NF-κB에 의존하는 성인 신경 발생의 변조 및 분석을위한 방법

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

마우스 좌골 신경에 생체 전기 생리 측정에서

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

개발 마우스 배아의 피질 유사 분열의 라이브 영상

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

간질의 마우스 모델에서 열 유발 발작에 대한 행동 화면

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

단백질 분석을 위한 마우스 망막에서 광수용체 세포 구획의 단리를 위한 두 가지 박리 방법

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...