PV 5용 실시간 fMRI를 설정하기 위한 AFNI 스크립트와 소프트웨어 지원을 위한 AFNI 팀을 공유해 주신 D. Chen 박사와 C. Yen 박사에게 감사드립니다. 이 연구는 NIH Brain Initiative 자금 지원 (RF1NS113278-01, R01 MH111438-01) 및 Martinos Center, German Research Foundation (DFG) Yu215 / 3-1, BMBF 01GQ1702 및 Max Planck Society의 내부 자금에 대한 S10 기기 보조금 (S10 RR023009-01)의 지원을 받았습니다.

이 연구는 독일 동물 복지법 (TierSchG) 및 동물 복지 실험실 동물 조례 (TierSchVersV)에 따라 수행되었습니다. 여기에 설명 된 실험 프로토콜은 윤리위원회 (§15 TierSchG)에 의해 검토되었으며 주 당국 (Regierungspräsidium, Tübingen, Baden-Württemberg, Germany)에 의해 승인되었습니다.
1. 소형 동물 연구를 위한 BOLD-fMRI 실험 설정 준비
<ol><li>콘솔 소프트웨어를 켜서 이미징 파라미터를 제어하고 MRI 데이터를 수집합니다. 참고: 제안된 실시간 fMRI 설정은 AFNI의 이미지 처리 기능과 병행하여 콘솔 소프트웨어(버전 6)의 매크로 기능을 사용하여 구현됩니다.</li>
	<li>작업 공간 탐색기를 사용하여 MR 시퀀스(예: 위치, 로컬라이저, 이완 향상을 통한 신속한 획득(RARE) 및 3D 에코 평면 이미징(EPI))를 찾은 다음 스캔 목록에 끌어서 추가합니다. 참고: 위치 및 로컬라이저 시퀀스는 뇌의 관심 영역(ROI)을 식별하는 데 사용됩니다. RARE 시퀀스는 해부학 스캔에 사용됩니다. 3D EPI 시퀀스는 동적 BOLD 응답을 측정하는 데 사용됩니다.</li>
	<li>미리 정의된 매크로 스크립트 "Setup_rt3DEPI" 및 "Feed2AFNI_rt3DEPI"를 매크로 스크립트 경로에 배치합니다(예: "/opt/(PV 버전)/prog/curdir/(사용자 이름)/ParaVision/매크로"). "데이터 재구성" 사용자 인터페이스 메뉴에서 3D EPI 재구성 옵션인 "사전 이미지 시리즈 활동" 및 "매크로 실행"을 활성화한 다음 "스캔" 버튼을 클릭하기 전에 미리 정의된 매크로 스크립트 "Setup_rt3DEPI"를 연결합니다. 참고: 매크로 스크립트는 추가 파일에 포함되어 있습니다.</li>
	<li>실시간 BOLD-fMRI 분석 및 시각화를 위해 AFNI 소프트웨어를 설치합니다.</li>
</ol>2. 카테터 삽입 및 환기 수술
<ol><li>그림 1과 같이 인공 호흡기와 온도계, 혈압 및 호흡 기록과 같은 생리적 상태 모니터링 시스템을 설정하십시오. 인공 호흡기로 60 ± 1 호흡 / 분의 일정한 주파수를 설정하고 피드백 제어 세트가있는 MR 호환 가열 패드를 사용하여 온도를 37 ° C로 설정하십시오.</li>
	<li>유도를 위해 5 % 이소 플루 란이있는 챔버에서 성인 수컷 Sprague-Dawley 쥐 (300-600g)를 마취시키고 기화기에서 수술을 위해 2-2.5 % 이소 플루 란을 전달합니다. 뒷발을 꼬집고 금단 반응이 없는지 확인하여 마취 깊이를 확인하십시오.</li>
	<li>환기를 위해 14G 플라스틱 캐뉼러로 동물에게 삽관하십시오 (70 % 공기와 30 % 산소의 혼합물로 60 ± 1 회 호흡 / 분). 호기말 이산화탄소 (CO2)를 25 ± 5 mmHg29의 범위로 조정하십시오. 알림: 삽관은 fMRI 실험을 통해 적절한 CO2 수준을 유지하는 데 중요합니다.</li>
	<li>동물을 수술대에 앙와위 자세로 놓고 전기 면도기로 허벅지를 면도하십시오. 그런 다음 수술 용 가위로 면도 한 피부를 절개하십시오. 참고: 절개 부위의 길이는 길이 방향으로 약 1-2cm입니다.</li>
	<li>카테터 삽입을 위해 절개 된 부위 아래에서 대퇴 동맥과 정맥을 찾고 개별 대퇴 동맥과 정맥을 주변 조직과 분리하십시오.</li>
	<li>분리 된 대퇴 동맥의 한쪽을 외과 봉합사로 고정하고 다른 쪽은 마이크로 불독 집게로 잡습니다. 그런 다음 대퇴 동맥의 묶인 부위 사이에 작은 절개를하십시오.</li>
	<li>작은 절개를 통해 대퇴 동맥에 카테터를 삽입하고 카테터와 동맥을 수술 봉합사와 함께 묶습니다. 생리적 모니터링 시스템으로 동맥 혈압을 80-120 mmHg 범위로 지속적으로 모니터링하고 동맥혈 가스를 정기적으로 측정하여 스캔하는 동안 최소 90 mmHg의 pO 2 및 30-45 mmHg의 pCO2를 유지합니다. 알림: 이 카테터 삽입은 fMRI 실험 중 동맥 혈압을 모니터링하는 데 중요합니다.</li>
	<li>대퇴 정맥의 양쪽 끝을 실크 꼰 수술 봉합사로 고정하십시오. 그런 다음 대퇴 정맥의 묶인 부위 사이에 작은 절개를하십시오. 봉합을 사용하여 봉합을 수행하십시오. 참고: 봉합사의 크기는 약 1-2cm입니다.</li>
	<li>카테터를 대퇴 정맥에 삽입하십시오. 카테터와 정맥을 외과 봉합사와 함께 묶으십시오. 참고: 이 카테터 삽입은 정맥을 통해 알파-클로랄로스를 투여하고 fMRI 실험 중에 마취 수준을 조정하는 데 중요합니다. 동물이 잘 마취되지 않으면 자발적으로 호흡하기 시작합니다. 이 경우 호흡 운동 인공물을 피하기 위해 더 많은 알파-클로랄로스를 투여해야 합니다.</li>
	<li>면도 한 피부에 수술 절개를 봉합하십시오. 수술 절차가 완료되면 대퇴 정맥에 연결된 카테터를 통해 ~ 80mg / kg의 용량으로 알파-클로랄로스 볼루스를 주입하여 동물을 마취시키고 동시에 이소 플루 란 투여를 중단하십시오.</li>
</ol>3. MRI 스캐너 안에 동물 배치
<ol><li>2.10 단계가 완료되는 즉시 마취된 동물을 MRI 스캐너로 옮기고 맞춤형 크래들에 고정합니다.</li>
	<li>동물에 실시간 피드백 직장 온도계를 삽입하여 동물의 체온을 모니터링합니다. 온도를 조절하기 위해 동물의 몸통 아래에 가열 패드를 놓습니다. MRI 스캔 중에 체온을 37.0 ± 0.5 ° C로 유지하십시오.</li>
	<li>근육 이완제인 판쿠로늄(~2mg/kg/h)의 혼합물에 ~25mg/kg/h 용액으로 알파-클로랄로스를 지속적으로 전달하면서 동물을 마취된 상태로 유지하고 fMRI 이미지에서 모션 아티팩트를 줄입니다. 생리적 상태에 따라 약물의 양과 환기 속도를 조정하여 혈압과 호흡을 모니터링합니다.</li>
	<li>fMRI 실험 중 건조를 방지하기 위해 동물의 눈에 안과 용 연고를 투여하십시오. 머리 움직임 아티팩트를 피하기 위해 두 개의 이어 바로 동물의 머리를 안전하게 고정하십시오.</li>
	<li>트랜시버 표면 코일을 헤드에 고정합니다. MRI 측정 전에 코일을 헤드의 Larmor 주파수(예: 599T에서 14.1MHz)에 맞추고 일치시킵니다. 알림: 여기에서 직경 22mm의 코일은 쥐의 전체 뇌를 덮는 데 사용됩니다.</li>
	<li>한 쌍의 바늘 전극을 숫자 1과 4 사이의 앞발 피부에 삽입하고 수술 용 테이프로 고정하십시오. 그런 다음 자극 입력 케이블을 이들 전극(30)에 연결한 후 자극이 제대로 작동하는지 확인합니다.</li>
	<li>동물을 MRI 보어에 삽입하고 대략 등심에 놓습니다.</li>
</ol>4. 해부학적 MR 이미지 측정
<ol><li>기본 사용자 인터페이스에서 보정 메뉴 버튼을 클릭합니다. 조정 플랫폼 사용자 인터페이스에서 다음 항목을 클릭하여 MRI 시스템의 보정을 수행합니다(콘솔 소프트웨어의 도움말 메뉴 참조): 기본 공진 주파수 찾기, RF 펄스 전력 보정, 최적의 수신기 게인 설정, 심볼링을 위한 동물의 B0 맵 측정, 비국소화 자유 유도 감쇠(FID) 적분을 기반으로 글로벌 선형 심 실행. 참고: 이 단계는 2분 미만이 소요됩니다.</li>
	<li>"스캔" 버튼을 클릭하여 위치 시퀀스를 실행하여 MRI 구멍 안에 있는 동물의 머리 위치를 찾습니다. 헤드가 등중심에 있지 않으면 헤드가 등중심에 위치할 때까지 크래들을 앞뒤로 움직이면서 헤드 위치를 조정합니다.</li>
	<li>"스캔" 버튼을 클릭하여 로컬라이저 시퀀스를 실행하여 헤드에서 ROI를 식별합니다. Map Shim을 선택하고 로컬라이저 이미지에서 전체 브레인을 포함하도록 심 볼륨의 ROI를 정의한 다음 "Shim upto" 옵션을 사용하여 고차(예: 2차 또는 3차) 심밍을 실행하여 ROI에서 주 자기장(B0) 비균질성을 줄입니다. 참고: 고차 시밍은 EPI 시퀀스를 사용할 때 BOLD-fMRI 데이터의 품질을 개선하기 위한 중요한 단계입니다.</li>
	<li>"스캔" 버튼을 클릭하여 T2 가중 RARE 시퀀스를 실행하여 코로나 보기에서 뇌 전체를 덮는 해부학적 이미지를 획득합니다(예: 반복 시간(TR) 4000ms, 유효 에코 시간(TE) 36.1ms, 매트릭스 128 x 128, 시야각(FOV) 19.2x19.2mm2, 슬라이스 수32, 슬라이스 두께 0.3mm, 희귀 인자 8). 참고: 다음 실시간 fMRI 시각화 단계에서 해부학적 이미지는 3D EPI 이미지를 템플릿으로 등록하는 데 사용됩니다.</li>
</ol>5. 실시간 fMRI 소프트웨어 설정 및 fMRI 반응 시각화
<ol><li>터미널 창을 열고 다음 명령을 사용하여 실시간 AFNI 플러그인 경로로 이동합니다. CD / 홈 / (사용자 이름) / rt_afni 참고: AFNI 플러그인 스크립트 "afni_rt"는 보조 파일에 포함되어 있습니다.</li>
	<li>아래 명령과 옵션을 사용하여 실시간 플러그인으로 AFNI 소프트웨어를 실행하십시오. 아프니 -RT -예스트플러그아웃 -DAFNI_REALTIME_MP_HOST_PORT=로컬 호스트:(포트 번호) -DAFNI_REALTIME_Graph=실시간 -DAFNI_FIM_IDEAL=(패러다임) 참고 : 첫 번째 경우에는 코드를 통해 외부 프로그램이 AFNI와 데이터를 교환 할 수 있지만 두 번째 경우에는 실시간 플러그인이 사용자 정의 로컬 호스트 및 포트에 대한 TCP 소켓을 열려고 시도합니다. 세 번째 및 네 번째 경우에서, 코드는 실시간으로 fMRI 데이터의 시간 과정을 플로팅하고, 실시간 fMRI 데이터가 획득될 때 각각 fMRI 시간 과정에서 사용자 정의 패러다임의 시간 과정을 플로팅할 것이다. 자세한 내용은 https://afni.nimh.nih.gov/pub/dist/doc/program_help/README.environment.html 확인하십시오.</li>
	<li>다음 옵션과 함께 그림 2 와 같이 "Dimon" 명령을 사용하여 정의된 예정된 AFNI BRIK 파일을 모니터링합니다. Dimon -tr (TR of EPI) -nt (NEPI의 반복) -rt -종료 -infile_pattern 실시간*. 브릭 -file_type 아프니 참고 : "Dimon"은 실시간 플러그인을 실행하는 "-rt"및 "-infile_pattern (데이터 이름) 옵션을 사용하여 AFNI 이미지 파일의 실시간 수집을 모니터링하는 명령입니다. BRIK -file_type AFNI"를 사용하면 플러그인이 특정 BRIK 파일을 읽고 표시 및 포맷을 위해 AFNI로 보낼 수 있습니다. 자세한 내용은 https://afni.nimh.nih.gov/pub/dist/doc/program_help/Dimon.html 확인하십시오.</li>
	<li>다음 옵션과 함께 "pvcmd"명령을 사용하십시오. pvcmd -a JMacroManager JMMExecuteMacro -category $USER -macro Feed2AFNI_rt3DEPI 참고: 이 코드는 EPI 획득을 위해 "스캔" 버튼을 클릭한 직후 백그라운드 매크로 스크립트 "Feed2AFNI_rt3DEPI"를 실행하기 위해 매크로 스크립트 "Setup_rt3DEPI"에 있습니다.</li>
	<li>"exec pvcmd" 명령을 다음 옵션과 함께 사용하여 EPI 획득 파라미터를 가져옵니다. exec pvcmd -a ParxServer -r ParamGetValue -psid $ParSpaceId -param (EPI의 PVM 파라미터) -id 10 -args $AcqKey $ParSpaceId $ProcnoPath</li>
	<li>"exec to3d" 명령을 다음 옵션과 함께 사용하여 EPI 원시 데이터를 백그라운드 매크로 스크립트 "Feed2AFNI_rt3DEPI"에서 실시간으로 AFNI 파일로 변환합니다. exec to3d -omri -xFOV $FOV_X -yFOV $FOV_Y -zFOV $FOV_Z -prefix $LastVolName $ImgFormat$Path2dseq</li>
	<li>EPI 기하학적 정보가 해부학 방향과 일치하는지 확인하십시오. 참고: "to3d" AFNI 명령은 그림 2와 같이 각 3D 볼륨 데이터가 모든 단일 TR 후에 저장될 때마다 fMRI 원시 데이터를 하나의 AFNI BRIK 데이터로 변환하기 위해 시야(FOV) 및 매트릭스 크기와 같은 기하학적 정보와 함께 자동으로 실행됩니다. 이미지 방향은 "to3d"의 기하학적 정보 매개 변수로 변경할 수 있습니다. 자세한 내용은 https://afni.nimh.nih.gov/pub/dist/doc/program_help/to3d.html 확인하십시오.</li>
	<li>전기 자극 아이솔레이터를 켜고 자극 블록을 사용하여 유발된 fMRI 연구(예: 3Hz, 4s 펄스 폭 300us, 2.5mA)에 대해 전기 앞발 자극을 수행합니다. 참고: 여기서 블록 설계 패러다임은 10개의 사전 자극 스캔, 3개의 자극 스캔 및 12개의 자극 간 스캔(에포크당 15개의 스캔)으로 구성됩니다.</li>
	<li>BOLD-fMRI 연구를 위해 "스캔" 버튼을 클릭하여 T2* 가중 3D EPI 시퀀스를 실행합니다(예: TR/TE 1500/14ms, 매트릭스 64 x 64 x 32, FOV 19.2 x 19.2 x 9.6mm3, 분해능 300 x 300 x 300μm3). 참고: "스캔" 버튼을 클릭하자마자 사전 정의된 매크로 스크립트를 사용하여 원시 데이터를 실시간으로 모니터링하고 처리합니다. 하나의 AFNI BRIK 데이터 세트가 변환되면 3D EPI 이미지에 대한 복셀 단위 시간 코스 그래프가 AFNI 소프트웨어에 표시되고 모든 단일 TR에 대해 자동으로 업데이트됩니다.</li>
	<li>해부학적 RARE 이미지 위에 EPI 이미지를 오버레이하려면 5.6단계와 같이 "to3d" 명령을 사용하여 RARE 이미지를 AFNI BRIK 데이터 세트로 변환한 다음 다음 옵션과 함께 "align_epi_anat.py" AFNI 스크립트를 사용하여 EPI 이미지를 해부학적 이미지에 등록합니다. align_epi_anat.py -anat anatomy_template_al+orig -epi epi.$(epi data number)+orig -epi_base 1 -접미사 _volreg -rat_align -비용 LPA -epi2anat 참고: 자세한 내용은 https://afni.nimh.nih.gov/pub/dist/doc/program_help/ align_epi_anat.py.html 확인하십시오.</li>
	<li>BOLD 응답의 기능 맵을 처리하려면 다음 옵션과 함께 "3dDeconvolve" 명령을 사용하여 특정 자극 시계열이 있는 3D+시간 데이터셋의 디콘볼루션을 계산합니다. 3dDeconvolve -입력 (입력 파일 이름)+orig. -n처음 0 -polort 3 -num_stimts 1 -stim_times 1 (자극 패러다임 파일 이름) '블록(4,1)' -stim_label 앞발 1개 -선전 -파우트 -패루트 참고: 공간 스무딩 또는 시간 필터링과 같은 이미지 처리 단계가 사용자 지정된 AFNI 데이터 처리 스크립트에 통합되었습니다. 자세한 내용은 https://afni.nimh.nih.gov/afni/doc/help/3dDeconvolve.html 확인하십시오.</li>
	<li>BOLD 신호의 기능 맵을 시각화하려면 AFNI 소프트웨어에서 대화형 클러스터링을 사용하십시오. "오버레이 정의" 옵션을 열고 AFNI 사용자 인터페이스 메뉴에서 "클러스터" 기능을 사용합니다.</li>
	<li>마지막 fMRI 스캔 후 MRI 스캐너에서 동물을 꺼내 승인 된 프로토콜에 따라 안락사시킵니다. 참고: AFNI의 이미지 처리 기능과 최신 콘솔 소프트웨어의 매크로 기능을 사용하여 실시간 fMRI 데이터를 처리했습니다. 매크로 기능에 대한 자세한 정보와 설명은 콘솔 소프트웨어의 도움말 메뉴에서 찾을 수 있습니다. AFNI 소프트웨어는 NIMH-AFNI 웹 사이트를 통해 직접 다운로드 할 수있는 프리웨어입니다. AFNI와 콘솔 시스템 간의 연결을 구축하기 위한 관련 스크립트가 첨부되어 있습니다.</li>
</ol>

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Sascha Köhler는 Bruker BioSpin MRI GmbH의 직원입니다.

fMRI 신호의 실시간 모니터링은 실험자가 동물의 생리학을 조정하여 기능적 매핑을 최적화하는 데 도움이 됩니다. 깨어있는 동물의 운동 인공물과 마취 효과는 fMRI 신호의 가변성을 매개하는 주요 요인이며, 그 자체로 신호의 생물학적 해석을 혼란스럽게합니다 31,32,33,34,35,36,37,38...

기능적 자기 공명 영상(fMRI)은 뇌의 신경 활동과 관련된 혈 역학적 반응 1,2,3,4,5,6,7,8,9, 예를 들어 혈중 산소 수준 의존적(BOLD), 뇌 혈액량 및 유동 신호를 측정하는 비침습적 방법입니다. 동물 연구에서, 혈역학적 신호는 마취(10), 깨어 있는 동물(11)의 스트레스 레벨, 뿐만 아니라 잠재적인 비생리학적 인공물, 예를 들어, 심장 맥동 및 호흡 운동(12,13,14,15)에 의해 영향을 받을 수 있다. 과제 관련 및 휴지기 상태 기능 역학 및 연결성 맵핑(16,17,18,19)에 대한 fMRI 신호의 후향적 분석을 제공하기 위해 많은 후처리 방법이 개발되었지만, 동물 뇌(20)에서 실시간 뇌 기능 맵핑 솔루션 및 순간 판독을 제공하는 기술은 거의 없으며(대부분은 주로 인간 뇌 맵핑(21)에 사용됨). 22,23,24,25,26,27). 특히, 이러한 종류의 실시간 fMRI 맵핑 방법은 동물 연구에서 부족하다. 실시간 뇌 상태 의존적 생리적 단계를 조사하고 동물 뇌 기능 연구를 위한 실시간 바이오피드백 자극 패러다임을 제공하기 위해 fMRI 플랫폼을 설정해야 합니다.
본 연구에서는 MRI 콘솔 소프트웨어의 맞춤형 매크로 기능을 사용한 실시간 fMRI 실험 설정을 설명하여 마취된 쥐의 일차 전발 체성 감각 피질(FP-S1)에서 유발된 BOLD-fMRI 반응의 실시간 모니터링을 시연합니다. 이 실시간 설정을 통해 기존 신경 이미지 분석 소프트웨어인 AFNI(Analysis of Functional NeuroImages)28을 사용하여 기능 맵에서 진행 중인 뇌 활성화와 복셀 방식으로 개별 시간 과정을 시각화할 수 있습니다. 동물 연구를 위한 실시간 fMRI 실험 셋업의 제조는 프로토콜에 기재되어 있다. 동물 설정 외에도 이미지 처리 스크립트와 병행하여 최신 콘솔 소프트웨어를 사용하여 실시간 fMRI 신호의 시각화 및 분석을 설정하는 자세한 절차를 제공합니다. 요약하면, 동물 연구를 위해 제안된 실시간 fMRI 설정은 MRI 콘솔 시스템을 사용하여 동물 뇌의 동적 fMRI 신호를 모니터링하기 위한 강력한 도구입니다.

<table>
	<tbody>
		<tr>
			<td>14.1T Bruker MRI system</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>A365 Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AcqKnowledge Software</td>
			<td>Biopac</td>
			<td>RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/</td>
			<td></td>
		</tr>
		<tr>
			<td>AFNI</td>
			<td>Cox, 1996</td>
			<td>RRID:SCR_005927, http://afni.nimh.nih.gov</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2SMO (ETCO2/SpO2 Monitor), Model 7100</td>
			<td>Novametrix Medical Systems Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP-Pharma</td>
			<td>Cat# 1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Master-9</td>
			<td>A.M.P.I</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoliter Injector</td>
			<td>World Precision Instruments</td>
			<td>Cat# NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancuronium Bromide</td>
			<td>Inresa Arzneimittel</td>
			<td>Cat# 34409.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ParaVision 6</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>RRID:SCR_001964</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>Cat# 10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat: Sprague Dawley rat</td>
			<td>Charles River Laboratories</td>
			<td>Crl:CD(SD)</td>
			<td></td>
		</tr>
		<tr>
			<td>SAR-830/AP Ventilator</td>
			<td>CWE</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-chloralose</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# C0128-25G;RRID</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time fmri brain mapping in animals

동적 fMRI 반응은 마취 상태 또는 깨어있는 상태의 동물의 생리적 조건에 따라 크게 다릅니다. 우리는 실험자가 획득 중에 fMRI 반응을 즉시 모니터링하도록 안내하는 실시간 fMRI 플랫폼을 개발했으며, 이는 동물의 뇌에서 원하는 혈역학 반응을 달성하기 위해 동물의 생리학을 수정하는 데 사용할 수 있습니다. 실시간 fMRI 설정은 14.1T 전임상 MRI 시스템을 기반으로 하며, 마취된 쥐의 일차 전발 체성 감각 피질(FP-S1)에서 동적 fMRI 반응의 실시간 매핑을 가능하게 합니다. 실시간 fMRI 플랫폼은 fMRI 신호의 가변성으로 이어지는 교란 소스를 조사하기 위한 후향적 분석 대신 맞춤형 매크로 기능과 MRI 시스템의 일반적인 신경 이미지 분석 소프트웨어를 사용하여 동적 fMRI 반응을 식별하는 보다 효과적인 체계를 제공합니다. 또한 동물의 뇌 기능 연구를 위한 즉각적인 문제 해결 가능성과 실시간 바이오피드백 자극 패러다임을 제공합니다.

그림 3 및 그림 4는 대표적인 실시간 복셀별 BOLD-fMRI 시간 과정 및 전기 앞발 자극(3Hz, 4초, 펄스 폭 300us, 2.5mA)을 사용한 기능 맵을 보여줍니다. fMRI 설계 패러다임은 10개의 사전 자극 스캔, 3개의 자극 스캔 및 총 8개의 에포크(130개 스캔)가 있는 12개의 자극 간 스캔으로 구성됩니다. 총 스캔 시간은 3분 15초(195초)입니다. 도 3은 블록-?...

Watch this Scientific Journal Video about Real-Time fMRI Brain Mapping in Animals at JoVE.com

Real-Time fMRI Brain Mapping in Animals

동물의 뇌 기능 매핑은 실시간 기능 자기 공명 영상 (fMRI) 실험 설정의 이점을 누릴 수 있습니다. 동물 MRI 시스템에 구현된 최신 소프트웨어를 사용하여 소형 동물 fMRI에 대한 실시간 모니터링 플랫폼을 구축했습니다.

동물의 실시간 fMRI 뇌 매핑

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Research

JoVE Journal

Neuroscience

3.4K 조회수.  Max Planck Institute for Biological Cybernetics. 동물의 뇌 기능 매핑은 실시간 기능 자기 공명 영상 (fMRI) 실험 설정의 이점을 누릴 수 있습니다. 동물 MRI 시스템에 구현된 최신 소프트웨어를 사용하여 소형 동물 fMRI에 대한 실시간 모니터링 플랫폼을 구축했습니다.

비디오: Real-Time fMRI Brain Mapping in Animals

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

쥐 두뇌에 동시 fMRI와 전기 생리학

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

연구

신경과학

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

기본 모드 네트워크를 검사하기 위해 Transcranial 자석 자극과 fMRI를 결합

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

Neuroscientific 연구 및 실시간 기능성 두피 매핑을위한 인적 Electrocorticographic (ECoG) 신호를 녹음하기

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

작은 동물의 만성 신경 레코딩을위한 초경량 동력 Microdrive의 설계 및 조립

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

하이 필드의 fMRI를 사용하여 성인 고양이의 청각 피질의 기능적 이미징

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

설치류에 동시 fMRI를 깊은 뇌 자극

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

관리의 실시간 fMRI Neurofeedback 훈련에 대 한 프로토콜

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

뇌의 세포 외 공간의 부피비와 비틀림을 정량화하기위한 테트라 메틸 암모늄을 이용한 실시간 이온 도입

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

라이브 셀 측정의 Odorant 수용 체 활성화를 사용 하 여 분석 결과 실시간 캠프

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...