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

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

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

프로 그레시브 - 비율 쥐의 Palatable 높은 지방과 높은 설탕 음식에 대한 응답

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

연구

신경과학

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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 ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

마우스의 전기 생리 녹음을위한 설계 및 초경량 무게의 제작, 조정 가능한 다중 전극 프로브

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

배아 마우스의 뇌에 추적 조건부 유전 Transsynaptic

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

MALDI 이미징 질량 분석에 대한 쥐 뇌 조직에 강글리오사이드의 감지 및 시각화를위한 DAN 매트릭스의 승화

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

대뇌 혈류와의 광 산란 특성을 동시에 평가<em&gt; 생체</em&gt; 다중 스펙트럼 확산 반사 이미징을 이용한 쥐 뇌

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

배아 운동 뉴런의 Axonal 투영 패턴의 시각화<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.