Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
Uploaded: 2018-07-10T15:00:00.000+00:00
Duration: 4 min 48 s
Description: 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

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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P., 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=5-Phosphonomethylquinoxalinediones+as+competitive+NMDA+receptor+antagonists+with+a+preference+for+the+human+1A/2A,+rather+than+1A/2B+receptor+composition.">5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition.</a> Bioorganic and Medicinal Chemistry Letters. 12 (7), 1099-1102 (2002).</li><li>Danysz, W., Parsons, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycine+and+N-methyl-D-aspartate+receptors:+physiological+significance+and+possible+therapeutic+applications.">Glycine and N-methyl-D-aspartate receptors: physiological significance and possible therapeutic applications.</a> Pharmacological Reviews. 50 (4), 597-664 (1998).</li><li>Liu, M. 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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9.2K 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

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

유전자 인코딩 된 센서를 사용 글루타민 유출 영상

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

연구

생체공학

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

다중 광자 세포 내 나트륨 이미징 CA1 피라미드 뉴런의 글루타메이트의 UV 매개 초점 Uncaging과 결합

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

신경과학

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

재조합 G 단백질 결합 수용체의 고처리량 스크리닝을 위한 "이중 첨가" 칼슘 형광 분석

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

생화학

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

효소 기반의 바이오 센서를 사용하면 알츠하이머 병 마우스 모델에서 토닉과 Phasic이 품목을 측정하는

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

AMPA 모니터링을 위한 하이 콘텐츠 분석 결과 수용 체 매매

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

해리 된 기본 해 마 문화에서 조미료 수용 체 인신 매매를 연구 하는 항 체 먹이 접근

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

정상 및 헌팅턴 마우스의 급성 뇌 슬라이스에서 글루타메이트 방출 및 섭취량의 단일 시냅스 지표

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

기본 마우스 중뇌 뉴런에서 자발적인 Ca2+ 플럭스와 다운스트림 효과를 정량화

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

One-channel Cell-attached Patch-clamp Recording

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

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

1 채널의 세포 부착 된 패치 클램프 녹음

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

생물학

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

근위축성 측삭 경화증 모델에서 시냅스 기능의 실시간 형광 측정

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...