이 연구는 국립 자연 과학 재단의 중국 (프로젝트 코드 번호 81373557, no. 81403327)에 의해 투자 되었다.

이 연구 윤리 위원회에는 중국의 중국 의학 과학원 (참조 번호 20160014)에 의해 승인 되었다. 모든 절차는 관리 및 실험 동물 사용 (국가 아카데미 압박, 워싱턴, D.C., 1996)에 대 한 국가 학회 건강 가이드에 따라 실시 했다. 4 성인 남성 쥐 (체중 250-280 g)이이 연구에 사용 되었다. 모든 동물에 12 h 명암 주기 제어 온도와 습도, 그리고 음식과 물에 자유 접근. 악기 및 현재 연구에 사용 된 자료 그림 1에 보였다 했다.  수술을 하기 전에 모든 장비 stereotaxic 프레임 및 유리 피 펫, 70% 에탄올을 사용 하 여 청소 했다.
1. 수술 절차
<ol><li>VPM stereotaxic 아틀라스9 (그림 2A)를 사용 하 여에 대 한 관심의 좌표 위치를 결정 합니다.</li>
	<li>1 µ L 마이크로-주사기 (약 10-20 µ m의 팁 직경)와 유리 제 micropipette 장착 준비 (그림 1D) 액체 파라핀으로 그것을 테스트.</li>
	<li>복 주입에 의해 7 %chloral 하이드 레이트 (0.7 mL/100 g)와 쥐를 anesthetize.</li>
	<li>깊은 마 취 확인 꼬리 핀치 철수 반사 페달, 전기 면도기와 동물의 머리의 면도 하 고 뒤 70% 에탄올에 각각 10 %povidone 요오드와 수술 부위의 3 번 스크럽.</li>
	<li>쥐 stereotaxic 장치에 무딘 귀 바 귀에 배치 하 여 고 입 홀더 (그림 1E)로 쥐의 위 앞 니를 장소 놓고 눈에 안과 연 고를 적용 합니다.</li>
	<li>70% 에탄올을 사용 하 여 다시 수술 부위의 머리 피부를 청소. 무 균 조건 하에서 수술을 유지 하기 위해 무 균 수술 장갑과 수건을 사용 합니다.</li>
	<li>화살 봉합 (그림 1 층)에 따라 메스로 피부에 절 개를 화살을 확인 합니다.</li>
	<li>근육과 살 균 면 밀고 도포를 사용 하 여 제어 출혈 (그림 1 층)을 수술을 통해 두개골에서과 다쳤어요.</li>
	<li>아틀라스 (그림 2A)에서 미리 정의 된 좌표를 사용 하 여 craniotomy (그림 1G)의 위치 (-3.3 m m Bregma 포인트, 2.6 m m 오른쪽 중간에) 결정 합니다.</li>
	<li>Craniotomy 라운드 팁 비트 (#106) (그림 1 H)와 함께 버 드릴을 사용 하 여 수행 하 고 계속 meninges (그림 1I)에 도달할 때까지 몇 분 이내는 1 mm 깊이 대 한 드릴.</li>
	<li>Dura mater 사출 사이트 (그림 1I)을 통해 대뇌 피 질을 노출 하는 microforceps를 사용 하 여 삭제하시오</li>
	<li>10 %BDA (10000 분자량, 증류수에) 솔루션 (그림 1J) 마이크로 주사기에 액체 파라핀을 변경 합니다.</li>
	<li>Microinjection 장치에 주사기를 탑재 하 고 마이크로 펌프 (그림 1 K)와 연결 합니다. 참고: 볼륨 주입 마이크로 펌프의 속도에 따라 달라 집니다. 여기 그것은 nL/30min (그림 1 L)에 조정 되었다.</li>
	<li>stereomicroscope에서 5.8 m m (그림 1 M)의 깊이에서 뇌의 대뇌 피 질의 표면을 통해 VPM에 유리 제 micropipette microinjection 장치를 수동으로 삽입 합니다.</li>
	<li>압력 주입 100 10%의 nL 마이크로 펌프 (그림 1 L, M)와 3 분 (35 nL/min)의 기간 동안 VPM에 BDA.</li>
	<li>주입 후 추가 5 분을 위한 장소에 피펫으로 유지 하 고 천천히 철수 다음.</li>
	<li>무 균 실 (그림 1N) 상처 봉합 사전 및 수술 후 진통에 대 한 당신의 현지 동물 보호 위원회 지침을 따릅니다.</li>
	<li>의식 회복은 완 치 될 때까지 따뜻한 복구 영역에 쥐를 놓습니다.</li>
	<li>그것의 감 금 소를 다시 복구 된 쥐를 반환 합니다.</li>
</ol>2. perfusions 및 섹션
<ol><li>일반적으로 10 일의 생존 시간 후 안락사를 유도 복 주입 하 여 사용 10% 우 레 탄 (2 mL/100 g)의 과다와 동물을 주입.</li>
	<li>(그림 1O) 후드에서 실험 쥐 perfuse<ol>
<li>호흡 중지가 위, 집게를 사용 하 여 마음에 액세스 하는 쥐의 흉 강을 엽니다. 대동맥으로 좌 심 실에는 정 맥 카 테 터를 삽입 한 다음 오른쪽 auricle 엽니다.</li>
		<li>먼저 perfuse 0.9% 인산 염 버퍼 식 염 수 (PBS) 생리 적인 온도 (37 ° C)와 약 1-2 분 혈액에서에서 종료 될 때까지의 선택을 취소 한 다음 0.1 M 인산 버퍼 (PB, pH 7.4)에 250-300 mL 4 %paraformaldehyde 계속.</li>
	</ol>
</li>
	<li>관류, 후 머리 피부를 incise 하 고, 두개골을 열고 쥐 뇌를 해 부. 후 해 부 뇌 실내 온도 (26 ℃), 0.1 m M PBS (pH 7.4) 두뇌 솔루션 (그림 1 P)에 몰입 될 때까지 4 ° C에서 3 일 동안에 30% 자당 다음 cryoprotect에서 2 h 4 %paraformaldehyde 수정.</li>
	<li>두뇌는 솔루션에 몰입은, 일단 뇌 매트릭스 (그림 1 분기)에 코로나 방향에 3 개의 블록으로 두뇌를 나눕니다. 중앙 블록 VPM와 s 1에 포함 되어 있습니다.</li>
	<li>톰 시스템 코로나 방향으로 슬라이딩 동결 단계에 40 µ m에서 뇌의 중앙 블록을 잘라. 이러한 섹션 (인물 1R, S) 0.1 M PBS (pH 7.4)를 가진 6-잘 접시에 질서를 수집 합니다.</li>
</ol>3. 표준 ABC 얼룩
참고: 두뇌의 모든 세 번째 코로나 섹션에서 무료 부동 섹션 표준 ABC 절차10BDA 라벨 시각화 사용 되었다.
<ol><li>약 1 분 동안 0.1 M PBS에 섹션 린스.</li>
	<li>(PH 7.4) 포함 0.3%에서 0.1 M PB 1 %ABC 솔루션에서 섹션을 품 어 실 온에서 1 h에 대 한 트라이 톤 X-100.</li>
	<li>워시 50 m m Tris 버퍼 (pH 7.4)에 세 번 섹션.</li>
	<li>0.02 %3, 3'-diaminobenzidine tetrahydrochloride (한 덩어리) 및 실 온에서 약 2-5 분 50 mM Tris 버퍼에 0.01% H2O2 를 포함 하는 솔루션에 섹션을 얼룩.</li>
	<li>워시 50 m m Tris 버퍼 (pH 7.4)에 세 번 섹션.</li>
	<li>현미경 슬라이드 표준 조직화 학적인 기법 (1T 그림; 참조 추가 비디오 파일 III, 관류 및 섹션)를 사용 하 여 섹션을 탑재 합니다.</li>
	<li>건조 실 온에서 하룻밤 공기에 섹션.</li>
	<li>탈수 알코올 (50%, 70%, 95%, 100%) 솔루션의 일련에 간단히 섹션. 약 15 각 솔루션에 슬라이드 잠수함 s. 각 단계 사이 건조 슬라이드를 허용 하지 않습니다.</li>
	<li>크 실 렌에서 섹션 3 회, 약 20 분을 선택을 취소 합니다.</li>
	<li>조각에 봉 선화의 2 또는 3 방울을 넣어 다음 coverslips 섹션에 배치 합니다.</li>
</ol>4. BDA 및 대뇌 피 질에 기본 신경 요소 더블 형광 성 얼룩
참고: 반면, 이중 형광 얼룩 실시 BDA 라벨의 상관 관계를 관찰에 대 한 위의 인접 섹션에 기본 신경 요소 streptavidin AF594와 함께 사용 하 고 형광 Nissl 얼룩 AF500/525와 counterstained.
<ol><li>약 1 분 동안 0.1 M PBS에 섹션 린스.</li>
	<li>(PH 7.4) 포함 0.3 %streptavidin-AF594 (1: 500) 및 0.1 M PBS에 AF500/525 녹색 형광 Nissl 얼룩 (1:1, 000)의 혼합 솔루션에서 섹션을 품 어 실 온에서 2 h에 대 한 트라이 톤 X-100.</li>
	<li>0.1 m M PB (pH 7.4)에 세 번 섹션을 씻어.</li>
	<li>현미경 슬라이드 표준 조직화 학적인 기술을 사용 하 여 섹션을 탑재 합니다. 약 1 h 공기에 섹션을 건조.</li>
	<li>관찰 하기 전에 증류수에 50% 글 리세 린과 형광 섹션에 coverslips를 적용 합니다.</li>
</ol>5. 더블 형광 얼룩 BDA 및 대뇌 피 질에서 수
참고: 이중 형광 얼룩 BDA 라벨 및 streptavidin AF594와 태양광 발전-immunochemistry 대뇌 피 질의 대상에서 대표 섹션에 수의 상관 관계를 관찰 실시 했다.
<ol><li>약 1 분 동안 0.1 M PBS (pH 7.4)에 대표 섹션 린스.</li>
	<li>차단 솔루션 3% 정상 염소 혈 청 0.3%를 포함 하는 섹션을 품 어 30 분 0.1 M PBS에 트라이 톤 X-100.</li>
	<li>전송 섹션 마우스 단일 클로 널 안티-PV IgG (1:1, 000) 0.1 M PBS (pH 7.4) 포함 1% 정상 염소 혈 청에 0.3%의 솔루션으로 4 ° c.에 하룻밤을 위해 트라이 톤 X-100</li>
	<li>다음 날, 0.1 m M PBS (pH 7.4)에 세 번 섹션을 씻어.</li>
	<li>염소 안티 mouse AF488 이차 항 체 (1: 500), 0.1 m M PBS (pH 7.4) 포함 1% 정상 염소 혈 청에서 streptavidin-AF594 (1: 500), 그리고 4' 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:40, 000) 및 0.3의 혼합 솔루션 섹션 노출 % 트리톤 1 h X-100입니다.</li>
	<li>4.5 4.3 단계를 반복 합니다.</li>
</ol>6입니다. 관찰
<ol><li>VPM, 축 삭 thalamocortical, 및 corticothalamic 신경의 이미지를 가져가 라.<ol>
<li>목표 렌즈를 장착 하는 confocal 이미징 시스템으로 형광 샘플 관찰 (4 x, 나: 0.13; 10 x, 나: 0.40;와 40 x, 나: 0.95). 사용 하 여 405 (파랑), 488 (녹색)의 여기 및 방출 파장 및 559 (빨간색) nm. 참고: 여기, confocal pinhole 152 µ m (4 배, 10 배)와 105 µ m (40 x)입니다. 이미지 캡처의 공간 해상도 1024 × 1024 픽셀 (4 배, 10 배) 및 640 × 640 픽셀 (40 x).</li>
		<li>40 µ m (Z 시리즈)의 두께에 각 섹션에서 2 µ m의 연속 프레임에 20 이미지를 가져가 라.</li>
		<li>다음과 같이 3 차원 분석을 위한 소프트웨어 시스템을 처리 하는 confocal 이미지와 단일-초점 이미지에 이미지를 통합: 설정된 시작 초점 평면 → 설정된 끝 초점면 → 설정된 단계 크기 → 선택 깊이 패턴 → 이미지 캡처 → Z 시리즈.</li>
	</ol>
</li>
	<li>디지털 카메라를 갖춘 가벼운 현미경에 의해 명시 이미지를 받아 (4 x, 나: 0.13; 10 x, 나: 0.40;와 40 x, 나: 0.95 렌즈). 노출 시간의 500 양 사용 사진 편집 소프트웨어를 사용 하 여 이미지의 명암과 밝기를 조정 하 고 레이블 추가를.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+precision+in+the+thalamic+projection+to+neonatal+mouse+barrel+cortex.">Topological precision in the thalamic projection to neonatal mouse barrel cortex.</a> J Neurosci. 15, 549-561 (1995).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (1998).</li><li>Davidoff, M., Schulze, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+avidin-biotin-peroxidase+complex+(ABC)+staining+combination+of+the+peroxidase+anti-peroxidase+(PAP)-and+avidin-biotin-peroxidase+complex+(ABC)-techniques:+an+amplification+alternative+in+immunocytochemical+staining.">Standard avidin-biotin-peroxidase complex (ABC) staining combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.</a> Histochemistry. 93, 531-536 (1990).</li><li>Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+axonal+diffusion+of+3000+molecular+weight+dextran+amines.">Fast axonal diffusion of 3000 molecular weight dextran amines.</a> J Neurosci Methods. 50, 95-103 (1993).</li><li>Kaneko, T., Saeki, K., Lee, T., Mizuno, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retrograde+axonal+transport+and+subsequent+visualization+of+tetramethylrhodamine+(TMR)+-dextran+amine+by+means+of+an+acidic+injection+vehicle+and+antibodies+against+TMR.">Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR.</a> J Neurosci Methods. 65, 157-165 (1996).</li><li>Medina, L., Reiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efferent+projections+of+the+dorsal+and+ventral+pallidal+parts+of+the+pigeon+basal+ganglia,+studied+with+biotinylated+dextran+amine.">The efferent projections of the dorsal and ventral pallidal parts of the pigeon basal ganglia, studied with biotinylated dextran amine.</a> Neuroscience. 81, 773-802 (1997).</li><li>DE Venecia, R. K., Smelser, C. B., McMullen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin+is+expressed+in+a+reciprocal+circuit+linking+the+medial+geniculate+body+and+auditory+neocortex+in+the+rabbit.">Parvalbumin is expressed in a reciprocal circuit linking the medial geniculate body and auditory neocortex in the rabbit.</a> J Comp Neurol. 400, 349-362 (1998).</li><li>Ojima, H., Takayanagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+two+anterograde+axon+tracers+to+label+distinct+cortical+neuronal+populations+located+in+close+proximity.">Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity.</a> J Neurosci Methods. 104, 177-182 (2001).</li><li>Kobbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog Neurobiol. 62, 327-351 (2000).</li><li>Vercelli, A., Repici, M., Garbossa, D., Grimaldi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+techniques+for+tracing+pathways+in+the+central+nervous+system+of+developing+and+adult+mammals.">Recent techniques for tracing pathways in the central nervous system of developing and adult mammals.</a> Brain Res Bull. 51, 11-28 (2000).</li><li>Liao, C. C., Reed, J. L., Kaas, J. H., Qi, H. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracortical+connections+are+altered+after+long-standing+deprivation+of+dorsal+column+inputs+in+the+hand+region+of+area+3b+in+squirrel+monkeys.">Intracortical connections are altered after long-standing deprivation of dorsal column inputs in the hand region of area 3b in squirrel monkeys.</a> J Comp Neurol. 524, 1494-1526 (2016).</li></ol>

저자는 공개 없다.

적절 한 추적 프로그램을 선택 하는 성공적인 신경 추적 실험에 대 한 중요 한 단계입니다. BDA, 높은 분자량 BDA의 가족에서 (10, 000 분자량) 저 분자 무게 BDA (분자량 3000)2,3 달리 참가자 신경 통로 통해 우선적으로 수송 될 추천 , 11 , 12 , 그러나 13., 많은 연구는 또한 그 높은 분...

높은 분자량 BDA (10000 분자량), 매우 민감한 트레이 서 20 년 이상의1중추 신 경계에서에서 신경 경로 추적 사용 되었습니다. BDA의 사용은 일반적인 신경로 추적 기법, BDA 라벨의 품질 다양 한 요인1,,23에 의해 동물에서 달라질 수 있습니다. 우리의 최근 연구 표시 BDA 라벨의 최적의 구조는 관련 된 적절 한 후 주입 생존 시간 뿐만 아니라 또한 착 색 방법4와 상관. 지금, 기존의 avidin 비타민 b 복합체 과산화 효소 복합물 (ABC), streptavidin fluorescein isothiocyanate streptavidin-AF594까지 착 방법은 이전의 연구2,3, BDA 라벨 공개에 대 한 사용 되었다 4,5. 비교에서는, 형광 성 얼룩 BDA에 대 한 수행할 수 있습니다 쉽게.
높은 분자량 BDA의 응용 프로그램을 확장 하기 위하여 세련 된 프로토콜은 현재 연구에 도입 되었다. 쥐 뇌에서 시상의 VPM에 BDA의 주입, 따라 이중 형광 얼룩, BDA 라벨의 상관 관계를 관찰을 위해 실시 되었다 뿐만 아니라 표준 ABC 얼룩의 일반적인 방법으로 계시 고 기본 했다 BDA 라벨 신경 요소 또는 streptavidin AF594 및 형광 Nissl histochemistry 또는 PV-immunochemistry, 대뇌 피 질의 대상에 수 각각. VPM 기본 somatosensory 피 (S1)6,,78사이 상호 신경 경로 통해 우리는 BDA 예상 thalamocortical 축 삭 및 corticothalamic에 라벨에 우리의 관찰 집중 s 1에서 예상된 셀 somas입니다. 이 과정에 의해 우리는 높은 분자 무게 BDA, 신경 라벨의 높은 품질을 얻기 위해 상세한 프로토콜 뿐만 아니라 다른 형광 신경 마커 및 형광 BDA 라벨의 조합에 세련 된 프로토콜을 제공할 것으로 예상 histochemistry 또는 immunochemistry입니다. 이 접근은 로컬 신경 회로 confocal 레이저 스캐닝 현미경 아래에서 그들의 화학 특성을 공부 하는 것이 좋습니다.

<table><tbody><tr><td>Biotinylated dextran amine (BDA)</td><td>Molecular Probes</td><td>D1956</td><td>10,000 molecular weight</td></tr><tr><td>Streptavidin-Alexa Fluor 594</td><td>Molecular Probes</td><td>S32356</td><td>Protect from light</td></tr><tr><td>500/525 green fluorescent Nissl stain</td><td>Molecular Probes</td><td>N21480</td><td>Protect from light</td></tr><tr><td>Brain stereotaxis instrument</td><td>Narishige</td><td>SR-50</td><td></td></tr><tr><td>Freezing microtome</td><td>Thermo</td><td>Microm International GmbH</td><td></td></tr><tr><td>Confocal imaging</td><td>Olympus</td><td>FV1200</td><td></td></tr><tr><td>system</td><td></td><td></td><td></td></tr><tr><td>Micro Drill</td><td>Saeyang Microtech</td><td>Marathon-N7</td><td></td></tr><tr><td>Sprague Dawley</td><td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td><td>SCKX (JUN) 2012-004</td><td></td></tr><tr><td>Vectastain ABC Kit</td><td>Vector Laboratories</td><td>PK-4000</td><td></td></tr><tr><td>superfrost plus microscope slides</td><td>Thermo</td><td>#4951PLUS-001</td><td>25x75x1mm</td></tr><tr><td>Photoshop and Illustration</td><td>Adobe</td><td>CS5</td><td></td></tr></tbody></table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

높은 분자량 biotinylated dextran 아민 (BDA)은 많은 십 년간 동안 매우 민감한 신경 해부학 추적 프로그램으로 사용 되었습니다. 이후 그 라벨의 품질은 여기, 다양 한 요인에 의해 영향을 제공 세련 된 프로토콜 높은 분자량 BDA의 응용 프로그램에 대 한 중앙 신경 시스템에 최적의 신경 라벨을 공부 합니다. 복 부 posteromedial 핵 (VPM)의 섬세 한 유리 피 펫을 통해 쥐에 시상에 BDA의 정위 적 주입 후 BDA 형광 streptavidin-알 렉 사 (AF) 594로 얼룩진 고 형광 Nissl 얼룩 AF500/525와 counterstained. 녹색 Nissl 얼룩의 배경, 빨간 BDA 라벨, 신경 셀 시체 및 axonal 터미널을 포함 하 여 더 분명히 somatosensory 피 질에서 시연 했다. 또한, BDA에 대 한 형광 염색을 두 번 및 칼슘 묶는 단백질 parvalbumin (PV) BDA 라벨 및 로컬 신경 공부 하는 기회를 제공 하는 대뇌 피 질의 대상에 태양광 발전-긍정적인 수의 상관 관계를 관찰 하기 위해 실시 되었다 회로 및 그들의 화학 특성 따라서,이 세련 된 방법은 고품질 신경 시상과 대뇌 피 질 사이의 상호 신경 경로 통해 높은 분자량 BDA로 머릿속에 적합 하지 않습니다 하지만 또한 동시 시범 허용 됩니다. 다른 신경 마커 형광 histochemistry 또는 immunochemistry입니다.

BDA는 VPM에 10 일 포스트 주입의 생존 강렬한 신경 라벨 해당 피 질 영역에 주입 쪽 (그림 2) 동측 생산에 대 한 충분 한 했다. 기존 ABC와 BDA에 대 한 절차를 얼룩이 지기 형광 공개 anterogradely를 포함 하 여 s 1에 신경 라벨의 비슷한 패턴 thalamocortical 축 삭을 표시 하 고 retrogradely corticothalamic 신경 (그림 2C, D를 표시 ).
<...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

여기, 선물이 효과적으로 상호 신경 통로 통해 방법 얼룩이 지기 형광으로 biotinylated dextran 아민 (BDA)를 공개 하는 세련 된 프로토콜. 그것은 미세 구조 분석 적합 BDA 라벨 및 다른 신경 요소 confocal 레이저 스캐닝 현미경 아래에서 구별의.

높은 분자량 Biotinylated Dextran 아민 Thalamocortical 프로젝션 쥐에 추적에 대 한 응용 프로그램 향상

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

improving-application-high-molecular-weight-biotinylated-dextran

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.9K 조회수.  China Academy of Chinese Medical Sciences. 여기, 선물이 효과적으로 상호 신경 통로 통해 방법 얼룩이 지기 형광으로 biotinylated dextran 아민 (BDA)를 공개 하는 세련 된 프로토콜. 그것은 미세 구조 분석 적합 BDA 라벨 및 다른 신경 요소 confocal 레이저 스캐닝 현미경 아래에서 구별의.

비디오: Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

신경 응답을 녹음하고 추적하는 주사에 대한 깨어 마우스의 작성

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

연구

신경과학

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

특정 기능 내장 신호를 결합하여 네크로의 마이크로 도메인과 투 광자 영상에서 뉴런의 대상 라벨링

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

조류 배아 Vestibulocochlear 신경 청각 섬유의 선택적 추적

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic는 콜레라 독소와 바이오틴 덱스 트란 아민 두 번 라벨로 이어 (僞) 바이러스에 주변 대상에서 추적

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

신생아 설치류의 복부 두개골에 개두를 확인하는 방법

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal Biocytin 레이블은 개별 두피 뉴런의 구조 - 기능 관계를 연구하는

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount 쥐의 망막에서 면역 조직 화학 및 칼슘 이미징 방법

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

레이저 유도 신경 세포의 추적에서 뇌 외식

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

해마의 생체 내 해부학 및 기능적 추적

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...