저자는이 연구 자금에 대 한 전체 게시물 학위 학자 프로그램 사무실과 노바 티 스 연구소 생물 의학 연구에 대 한 감사 하 고 싶습니다.

1입니다. 셀의 준비
참고:이 프로토콜을 데이터 생성을 포함 하 여 잠재 인코딩 NR1 및 NR2A 세포로 불리고 HEK293 세포를 사용 합니다.
<ol><li>HEK293 세포의 적절 한 수 씨 하 고 적절 한 최종 농도 (1.00 µ L 각)에서 NR1 / NR2A 바이러스를 추가 합니다. 384-잘 접시를 사용 하 여 10, 000 셀/잘 30 µ L의 최종 볼륨.</li>
	<li>또는 적절 한 휴대폰 번호와 (384-잘 접시, 10, 000 사용 하 여 셀/잘 30 µ L의 최종 볼륨에서)에 대 한 볼륨 HEK293 NR1 NR2A 셀 씨. NR2A 식 유도 및 복합 보호 추가 2 µ g/mL의 농도에서 항생물질을 추가 (100 µ M MDL105, 519 또는 5 µ M CGP07066724).</li>
	<li>참고: 인코딩 NR1, NR2 잠재 5 x 109 -10 x 109 밀리24당 감염 단위 사이 대략 titer가 있어야 합니다.</li>
</ol>2입니다. NMDA 수용 체 활동의 측정
<ol><li>HEK293 세포 중 보호 화합물 존재 NR1/NR2 또는 HEK293 NR1 NR2A 세포 transduced 384-잘 접시를 준비 합니다. 사용 하 여 명확한 바닥, 블랙 프레임, 폴 리-D-리 코팅된 판. 참고: 보호 100 µ M와 MDL105, 519 글리신, 5 µ M 동안에 감도 수 여 하는 CGP070667은 조미료에 감도 부여 하는 데 사용 됩니다.</li>
	<li>16 시간 (하룻밤) 37 ° C, 5% CO2 접시를 품 어.</li>
	<li>인큐베이터에서 접시를 제거 하 고 부드럽게 호환 유기화학 컨테이너 접시를 뒤집어 감속에 의해 셀 미디어를 삭제. 부드럽게 접시의 가장자리에서 미디어를 청소 하 고 나머지 미디어 배수를 종이 타월에 접시를 누릅니다.</li>
	<li>인큐베이션 버퍼 (HBSS pH 7.5, 20 mM HEPES, 1 mM MgCl2, 1mm probenecid) 10 mL에 calcium6 염료 (1 접시 크기)의 한 유리병 solubilizing로 calcium6-염료를 준비 합니다. 37 ° C, 5% CO2에서 2 시간 동안 접시를 품 어.</li>
	<li>인큐베이터에서 접시를 제거 하 고 10 분 동안 실내 온도에 조정 하는 셀을 하자.</li>
	<li>부드럽게 2.3 단계에서 설명한 대로 calcium6 염료를 쏟아 하 고 분석 결과 버퍼 (HBSS pH 7.5, 20 mM HEPES, 1.8 m m CaCl2, 1mm probenecid)의 30 µ L를 추가 합니다.</li>
	<li>3 세척의 2.6 두 번 총에 대 한 단계를 반복 합니다. 마지막 세척 후 분석 결과 버퍼의 30 µ L 셀에 둡니다.</li>
	<li>실 온에서 5 분간 휴식 하는 셀을 하자.</li>
	<li>400 µ M 글리신 및 400 µ M 조미료 (100 µ M의 최종 농도, 아래 설명 된 대로 결정 되는 ligands의 최적 농도) 4-fold 재고 하 여 리간드 접시를 준비 합니다. V-아래 384-잘 접시에 ligand 주식의 25 µ L의 최소 전송.</li>
	<li>Ligand 접시와 셀 플레이트 FDSS (기능성 약물 검사 시스템)로 로드 합니다.</li>
	<li>30 초 동안 셀 플레이트의 기준선 형광 (Fo)를 측정 합니다. 5 분 동안 calcium6 염료 형광을 기록 하 여 FDSS 분배 기능과 측정 칼슘 플럭스를 사용 하 여 셀 접시에 ligand의 10 µ L를 전송 합니다.</li>
	<li>기준선 형광 (F최대/Fo)에 의해 얻은 최대 형광을 나누어 최대한 형광 비율을 계산 합니다.</li>
</ol>3입니다. NMDAR 식 레벨의 최적화
<ol><li>사용 하 여 잠재의 최적 크기를 결정, NR1 및 NR2A 바이러스의 적정을 수행 합니다. HEK293 세포 (10, 000 셀/잘의 추천, 미디어의 30 µ L) 384-잘 접시를 준비 하 고 단계 2.1에에서 설명 된 대로 보호 화합물 포함.</li>
	<li>접시의 다른 행에 있는 NR1 바이러스의 다양 한 금액을 추가 (예: 행 A-E 0 µ L, 2 µ L, 1 µ L, 0.5 µ L, 0.25 µ L를 각각 추가). 데이터의 정확성에 대 한 중복을 추가 합니다.</li>
	<li>접시의 다른 열에 NR2A 바이러스의 다양 한 금액을 추가 (예: 행 A-E 0 µ L, 2 µ L, 1 µ L, 0.5 µ L, 0.25 µ L를 각각 추가). 데이터의 정확성에 대 한 중복을 추가 합니다.</li>
	<li>16 시간 (하룻밤) 37 ° C, 5% CO2 접시를 품 어.</li>
	<li>FDSS (위 참조)에 칼슘 플럭스 측정을 수행 하 여 최적의 비율과 NR1 및 NR2A 바이러스의 금액을 결정 합니다.</li>
</ol>4. 리간드 적정
<ol><li>2.3 단계에서 설명한 대로 셀을 준비 합니다.</li>
	<li>4-fold 재고 솔루션으로 다른 ligand의 농도 (100 µ M)를 흠뻑 젖 게 하는 면 전에 서 각 ligand의 희석을 준비 합니다. 예: 글리신의 10 µ M 최종 테스트 농도, 글리신 400 µ M 조미료 등의 40 µ M 재고 솔루션을 준비.</li>
	<li>FDSS 측정 단계 2.11에에서 설명 된 대로 진행 합니다.</li>
</ol>5. 복합 테스트
<ol><li>1 단계에서 설명한 대로 셀을 준비 합니다.</li>
	<li>분석 결과 버퍼에서 최종 ligand 농도의 5 재고 ligand 접시를 준비 합니다.</li>
	<li>4-fold 재고 분석 결과 버퍼에서 화합물의 원하는 최종 농도의 화합물 접시를 준비 합니다. 분석 결과 버퍼에 10 µ M의 최종 화합물 농도, 40 µ M 재고 솔루션을 준비 합니다.<ol>
<li>계속 디 메 틸 sulfoxide (DMSO)의 농도 상수 모든 샘플에서 분석 결과 버퍼에 더 희석 하기 전에 DMSO에 화합물의 사전 희석을 준비 하 여.</li>
		<li>최종 분석 결과 3 그리고 30 µ M 사이에서 화합물의 복용량 응답, 1에서 10 m m까지 DMSO에 화합물의 사전 희석을 준비 합니다. 4-fold 재고 농도 분석 결과 버퍼에 희석. DMSO의 최종 농도 0.5%를 넘지 말아야 한다. 참고: 그것은 triplicates에 각 샘플을 테스트 하 고 좋습니다.</li>
	</ol>
</li>
	<li>FDSS에 ligand, 화합물, 및 셀 접시를 로드 합니다.</li>
	<li>30 초 동안 기준선 형광을 측정 합니다.</li>
	<li>복합 주식의 10 µ L을 추가 하 고 5 분 동안 형광을 측정.</li>
	<li>Ligand 주식의 10 µ L을 추가 하 고 5 분 동안 형광을 측정.</li>
	<li>측정의 마지막 5 분 동안 형광 비율의 곡선 아래의 영역을 계산 하 여 형광 비율의 전체를 확인 합니다. 참고: 또는, ligand 추가 후 형광의 비율을 최대한 사용할 수 있습니다 분석을 위해.</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+II+Inhibitors+Can+Enhance+Baculovirus-Mediated+Gene+Expression+in+Mammalian+Cells+through+the+DNA+Damage+Response.">Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response.</a> International Journal of Molecular Science. 17 (6), (2016).</li></ol>

저자는 충돌의 관심 및 공개 아무것도 있습니다.

이 분석 결과의 성공 사용 HEK 세포의 건강에 따라 달라 집니다. 지 수 성장을 겪고 세포와 낮은 통로 번호와 함께 사용 해야 합니다. 이 분석 결과 많은 전송 고 추가 그렇게 주의 사용 하 여 솔루션의 결과에 더 높은 정확도 보장 합니다. 화합물 및 다른 모든 시 약의 농도 또한 오류를 최소화 하기 위해 크로스 체크 해야 합니다. 칼슘 유출 분석 결과 대 한 분석 결과 버퍼와 셀 미디어를 교체할 때 ?...

현재 의학의 발전, 수명 크게; 증가 했다 그러나, 그래서 나이 관련 된 질병의 유행이 하고있다. 질병 중앙 신경 조직 (CNS) 정신 분열 증, 루 경화 증 (ALS), 알 츠 하이 머 병, 파 킨 슨 병, 다른 사람, 사이 등의 예외가 있으며 향후 10 년간1, 증가 예상 2 , 3. N-메 틸-D-aspartate 수용 체 (NMDAR)로 알려진 ionotropic 글루타민 산 염 수용 체의 고장 Alzheimer의 질병, 정신 분열 증, 외상 성 뇌 손상, 뇌졸중, 당뇨병, 및 보증, 녹 내장에 연결 되어있다는 효과적인, 질병 수정 요법4,5,,67의 개발에 대 한 그들의 생물학을 공부 해야 합니다.
NMDARs는 4 개의 단위체 또는 소 단위4,,89의 구성 됩니다. NMDAR의 구조 구성 뇌7,10발달 및 지역 다양성을 보여줍니다. NMDARs는 시 냅 스가 소성, 인식, 그리고 리듬의 세대에서 호흡, 운동11,,1213에서 포함 된다. 주로 휴식 막 잠재력에 비 전도성은 전압-개폐 통로 (-70 mV) 마그네슘 이온의 투과 방지에 의해 차단 됩니다. 채널 두 ligands, 조미료 및 글리신/D-떠들고의 바인딩에 의해 활성화 되 고 시 냅 스 막에 동시 도발은 AMPA 수용 체, ionotropic 글루타민 산 염 수용 체의 다른 서브 클래스에 의해 중재. 도발은 양이온, 특히 칼슘14,,1516의 유입 활성화 NMDAR의 마그네슘 막힘을 제거 합니다. NMDAR 활성화 세포 생존을 위해 필수적 이다, 비록 과도 한 활성화 세포 죽음17,,1819 excitotoxicity 통해 발생할 수 있습니다. 수용 체의 복잡 한 자연 뿐만 아니라 게 연구를 수행 하기 위해 도전 효과적인 치료 개발에 필요한.
다른 메서드는 NMDAR 연구 개발 되었습니다. 그러나, 각자 주의 동반 했다. 예를 들어 하나의 널리 사용 기법 항생물질을 유도할 수 있는 발기인 (Tet에)20의 통제 안정적인 셀 라인에서 세포내 칼슘에서 NMDAR 중재 변화를 측정 하는 형광 기반 분석 결과 이다. 그러나이 시스템에 필요한 ligands의 supramaximal 농도 NMDAR 억제제는 측정 중에 존재 하는 요구는 그것 경쟁 길 항 근의 활동을 검출 하기 거의 불가능. 다른 유사한 시스템에서 기능 수용 체의 식이 발생 독성, 셀 문화를 보존 하기 위해 마 취 제21,22 같은 채널 차단제를 요구 합니다. 이러한 채널 차단제는 수용 체의 코어에 앉아서 그들은 수용 체의 기능 연구 방해 플레이트 기반 포맷에 (서) 특히, 밖으로 세척 하기 어려운. 마지막으로, 패치 죄 같은 electrophysiological 측정에서 제한 된 처리량, 그리고 대규모 연구는 매우 비싼23. 도 불구 하 고, 위의 시스템은 민감한 글리신 자극; 따라서, 글리신 종속 활동은 NMDAR의 공부는 도전 된다.
여기, 우리가 NMDAR 논의 한계를 극복 하는 공부에 대 한 새로운 접근 방식을 설명 합니다. 우리의 기술은 작은 16 시간에는 소 단위의 최적의 비율로 기능 수준에서 수용 체를 표현 하는 잠재 식 시스템에 대문자로 바꿉니다. 또한, 잠재를 사용 하 여 고유 재조합 NMDAR의 광범위 한 특성을 제공 하는 간단 하 고 조합 접근 수 있습니다. 다른 분석 실험, 달리이 프로토콜은 약한 길 항 제를 사용 하는 채널 차단제를 필요 하지 않습니다. 방법의 가장 강한 장점은 약한 길 항 근의 그 후 유실, 수용 체는 글리신/D-떠들고와 조미료의 듀얼 변조 뿐만 아니라 개별 글리신 및 글루타민 산 염 바인딩 사이트의 ligand 바인딩 사이트입니다. 분석 결과 NMDAR 수용 체의 알려진된 약리학 및 그것의 알려진된 양수와 음수 변조기의 효과. 마지막으로,이 체 외에서 세포 분석 결과의 발생 과도 한 칼슘 유입으로 인 한 세포 독성을 극복 하 고 NMDAR 변조기의 발견을 가속 수 있습니다 높은 처리량 패션에 수용 체의 기능 연구에 대 한 허용 에 질병 상태.

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

N-메 틸-D-aspartate (NMDA) 수용 체 (NMDAR) ionotropic 글루타민 산 염 수용 체로 분류 됩니다 있고 학습과 기억에 중요 한 역할. NMDAR 오작동, 표현으로 어느 이상-또는 아래-activity 돌연변이, 변경된 식, 인신 매매, 또는 지역화로 인 한 수많은 질병, 특히 중앙 신경 조직에에서 기여할 수 있다. 따라서, 화합물 및 작은 분자의 발견을 촉진 뿐만 아니라 이해 하는 수용 체의 생물학 신경 질환에 대처 하기 위해 지속적인 노력에서 결정적 이다. 수용 체를 공부 하 고 현재 접근 제한이 낮은 처리량, 높은 비용과 excitotoxicity NMDAR 중재를 방지 하기 위해 채널 차단제의 필요한 존재로 인해 그것의 기능적인 능력을 공부 하는 무 능력을 포함 하 여. 또한, 기존의 분석 결과 시스템만 조미료에 의해 자극에 민감한 고 글리신은 NMDAR의 다른 공동 ligand에 의해 자극 감도 부족. 여기, 선물이 공동 ligands, 조미료와 D-떠들고/글리신에 감도 NMDA 수용 체를 높은 처리량 힘을 가진 첫 번째 접시 기반 분석 결과. 이 이렇게 다른 NMDAR 소 단위 구성의 연구를 허용 하 고 글리신-또는 조미료에 민감한 모드에서 수용 체의 기능 연구를 허용. 또한, 방법 측정 동안 억제제의 존재를 필요 하지 않습니다. 이 분석 결과 함께 긍정적이 고 부정적인 allosteric 변조기의 효과 검출 될 수 있다 고 NMDAR의 알려진된 약리학 우리의 시스템에 복제 되었습니다. 이 기술은 기존 방법의 한계를 극복 하 고 비용 효율적입니다. 우리는이 새로운 기술은 NMDAR 중재 병 리에 대 한 치료의 발견을 가속화 될 것을 믿습니다.

작은 분자의 효과 테스트 하기 전에 하나 NMDARs 최적의 ligand 농도의 최적의 식 수준을 결정 해야 합니다. 설명, HEK293 세포 384-잘 접시에 잘 당 10000 셀에서 시드 했다 5 µ M의 CGP060667, 다음 불리고 NR1 및 NR2A의 다양 한 양의 바이러스. 인큐베이션 하룻밤, ligand 유도 된 칼슘 후 유량 측정 되었다 (그림 1). 이러한 실험의 결과 공개 최고의 수량 및 각 소 단위...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

글리신/D-떠들고와 조미료에 감도와 NMDA 수용 체를 공부 하는 높은 처리량 칼슘-플럭스 분석 결과

이 프로토콜의 목표는 더 큰 규모에 NMDA 수용 체 (NMDAR)의 연구를 촉진 하기 위하여 작은 분자의 modulatory 효력의 검사 및 치료 응용 프로그램입니다.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

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Neuroscience

9.1K 조회수.  Novartis Institutes for BioMedical Research. 이 프로토콜의 목표는 더 큰 규모에 NMDA 수용 체 (NMDAR)의 연구를 촉진 하기 위하여 작은 분자의 modulatory 효력의 검사 및 치료 응용 프로그램입니다.

비디오: A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

파리에서 에탄올 민감도를 측정하는 간단한 방법

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

연구

신경과학

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 ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

에 타고난 및 시간주기 규제 Photobehavior을 공부하는 빛 환경의 분석<em&gt; 초파리</em&gt; 애벌레

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

성상 세포 운송업자 전류의 디컨 볼 루션 분석 글루타민산 정리의 시간 과정을 도출

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

초점 방사선 억제와 성인 시상 하부의 신경 발생의 기능 심문

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em&gt; 노랑 초파리</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

상호 빛과 전자 현미경은 β - 아밀로이드 플라크와 소교의 상호 작용을 연구하는

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.