Name: construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo
Uploaded: 2016-05-12T13:00:00
Description: Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

우리는 전자 제품에 대한 지원은 G의 qi5 및 G QS5 cDNA를, A. 슈바이처를 제공 B. 콘 클린 (캘리포니아 대학 샌프란시스코)를 감사, N. 테일러 증거 읽기 클론, 이안 Glaaser 로버트 리프킨의 심사에 대한 지원 및 TN-XXL에 대한 올리비에 Griesbeck. (; DA037170 DA029706), 바이오 메디컬 이미징 및 생물의 국립 연구소 (NIBIB) (EB003832), 호프만 - 라 로슈 (88610A)과 &quot;신경 과학이 작품은 약물 남용에 미국 국립 연구소 (NIDA)를 통해 연구비에 의해 지원되었다 NIDA (DA007315)를 통해 남용 &quot;훈련 보조금의 마약 관련.

본 연구에서 수행되는 모든 동물의 절차는 기관 동물 관리 및 사용위원회 (IACUC) 지침에 따라, 그리고 시내산에서 의학의 아이칸 학교와 캘리포니아 대학 샌디에고에서 IACUCs에 의해 승인되었습니다. 1. HEK293 세포 변형에 대한의 Lentivirus를 GPCR이 발현 생성 <ol><li> 상용 소스, 예를 들어, cdna.org에서 특정 GPCR에 대한 cDNA를 가져옵니다. 다르게는, PCR을 이용하여 cDNA 라이브러리로부터 GPCR ?...

<ol>
	<li>Day, J. C., Kornecook, T. J., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+vivo.+microdialysis+to+the+study+of+cholinergic+systems.">Application of in vivo. microdialysis to the study of cholinergic systems.</a> Methods. 23, 21-39 (2001).</li><li>Robinson, D. L., Venton, B. J., Heien, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+subsecond+dopamine+release+with+fast-scan+cyclic+voltammetry+in+vivo.">Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo.</a> Clin Chem. 49, 1763-1773 (2003).</li><li>Liang, R., Broussard, G. J., Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Chemical+Neurotransmission+with+Genetically+Encoded+Fluorescent+Sensors.">Imaging Chemical Neurotransmission with Genetically Encoded Fluorescent Sensors.</a> ACS Chem Neurosci. , (2015).</li><li>Okubo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+extrasynaptic+glutamate+dynamics+in+the+brain.">Imaging extrasynaptic glutamate dynamics in the brain.</a> Proc. Natl. Acad. Sci. USA. 107, 6526-6531 (2010).</li><li>Marvin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+fluorescent+probe+for+visualizing+glutamate+neurotransmission.">An optimized fluorescent probe for visualizing glutamate neurotransmission.</a> Nat Methods. 10, 162-170 (2013).</li><li>Nguyen, Q. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+biosensor+for+neurotransmitter+release+and+in+situ+receptor+activity.">An in vivo biosensor for neurotransmitter release and in situ receptor activity.</a> Nat Neurosci. 13, 127-132 (2010).</li><li>Muller, A., Joseph, V., Slesinger, P. A., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+reporters+reveal+in+vivo+dynamics+of+dopamine+and+norepinephrine+release+in+murine+cortex.">Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex.</a> Nat Methods. 11, 1245-1252 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J Vis Exp. , e3998 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. , (2009).</li><li>Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., Bourne, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+three+amino+acids+switches+receptor+specificity+of+Gq&#945;+to+that+of+Gj&#945;.">Substitution of three amino acids switches receptor specificity of Gq&#945; to that of Gj&#945;.</a> Nature. 363, 274-276 (1993).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1, 3166-3173 (2006).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J Vis Exp. , (2012).</li><li>Drew, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nat Methods. 7, 981-984 (2010).</li><li>Shih, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> J Cereb Blood Flow Metab. 32, 1277-1309 (2012).</li><li>Yamauchi, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+ligand-gated+ion+channel+receptors+with+genetically+encoded+Ca2++sensors.">Characterizing ligand-gated ion channel receptors with genetically encoded Ca2+ sensors.</a> PLoS One. 6, e16519 (2011).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J. Neurosci. 32, 13819-13840 (2012).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Cui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+activation+of+striatal+direct+and+indirect+pathways+during+action+initiation.">Concurrent activation of striatal direct and indirect pathways during action initiation.</a> Nature. 494, 238-242 (2013).</li></ol>

CNiFERs의 생성은 광학적으로 생체 내에서 뇌의 신경 전달 물질의 방출을 측정하기위한 혁신적이고 독특한 전략을 제공합니다. CNiFERs 이상적으로 신경 전달 물질에 대한 예 extrasynaptic 해제, 볼륨 전도를 측정하기에 적합합니다. 중요한 것은, 각 CNiFER은 뇌의 신경 전달 물질의 농도의 변화 생리 광 측정을 제공하는 천연 GPCR의 특성을 가지고있다. 지금까지 CNiFERs은 아세틸 콜린을 검출 ?...

완전히 신경 뇌에서 통신하는 방법을 이해하기 위해, 생체 내에서 신경 전달 물질의 방출을 측정하는 방법을 갖는 것이 필요하다. 생체 내에서 신경 전달 물질을 측정하기위한 몇 가지 잘 확립 된 기술이있다. 하나의 일반적으로 사용되는 기술은 캐뉼라 뇌에 삽입 뇌척수액 소량의 수집 및 고성능 액체 크로마토 그래피 및 전기 검사 1을 사용하여 분석되는 미세 투석이다. 미세 투석 프로브 몇 직경 정도의 공간 분해능을 가지며, 예를 들어 ~ 200 μm의 마이크로 프로브 직경 0.5 mm. 이 기법의 시간 해상도는, 그러나, 일반적으로 ~ 5 분 1 이상을 지속 샘플링 간격으로 느립니다. 또한, 분석은 실시간으로 제작되지 않습니다. 다른 기술은 뇌에 삽입되는 탄소 섬유 프로브를 사용하여 고속 스캐닝 순환 전압 전류 법 (FSCV)이다. FSCV 우수한 온도가구강 해상도 (초 단위), 고감도 (나노 몰), 30 μm의 5의 프로브 직경 공간 해상도. 그러나 FSCV는 탄소 전위차 프로브 (2)에 전압 특성 산화 및 환원 프로파일을 생성 송신기들로 제한된다. 신경 전달 물질을 측정하는 세 번째 기술은 유 전적으로 인코딩 된 신경 전달 물질 (NT) 바이오 센서 (3)를 통해 직접입니다. 이 방법으로 융합 단백질은 형광 공명 에너지 전달 (FRET) - 기반 형광체 (4)의 쌍 또는 순열 GFP (5)에 결합 된 송신기에 대한 리간드 결합 도메인을 포함하는 생성된다. 앞의 두 방법과 달리,이 바이오 센서는 전적으로 인코딩 된 형질 전환 동물의 생산을 통해 또는 급성 세포 감염 바이러스 제제의 사용과 같은 신경 세포와 같은 숙주 세포의 표면에 발현. 현재까지 유전자 부호화 바이오 센서 만 detectin 개발되어왔다g 글루타메이트와 GABA 3-5. 이러한 기술에 대한 제한은 nM의 범위에서 낮은 감도 및 G 단백질 결합 수용체 (GPCR에)를 통해 신호를 예를 들어 송신기, 고전 신경 전달 물질, 신경 펩티드 및 신경 조절의 다수 검출 확대 없게되었다. 실제로, 인간 게놈에서 거의 800 GPCRs에있다. 이러한 부족을 해결하기 위해, 우리는 GPCR을 통해 신호를 어떤 신경 전달 물질의 광학적 측정 자료에 혁신적인 도구를 개발했습니다. CNiFERs (셀 기반의 신경 전달 물질의 형광 설계 기자)가 자극하면 [Ca를 2+ 즉이 유전자 부호화 FRET 계 칼슘 센서에 의해 검출되어, 세포 내에서의 증가를 유발 특정 GPCR을 발현하도록 유전자 클론 HEK293 세포이다 TN-XXL. 따라서 CNiFERs은 신경 전달 물질 수용체, 형광의 변화에​​ 직접 결합 실시간 광 (R)을 제공하는 변환지역의 신경 전달 물질 활동의 EAD 아웃. 특정 신경 전달 물질에 대한 고유 수용체를 이용하여, CNiFERs 화학 특이성, 친 화성 및 내생 발현 수용체의 시간적 동성을 보유한다. 지금까지, 우리는 CNiFERs 세 가지 유형, 도파민을 검출하는 M1 수용체를 이용하여 아세틸 콜린의 검출을 작성한 D2 수용체를 사용하고 α1a 수용체 6,7을 사용하여 노르 에피네프린을 검출하기위한 하나. CNiFER 기술은 GPCR의 유형은 의무가있어 쉽게 확장과 확장 성이다. 이 조브 기사에서, 우리는 설명하고 모든 응용 프로그램에 대한 생체 CNiFERs의 설계, 실현 방법, 테스트를 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">pCDH-CMV-MCS-EF1-Puro&#160;</td>
			<td style="width:191px;">System Biosciences</td>
			<td style="width:429px;">CD510B-1</td>
			<td style="width:207px;">Cloning: for generating lentivirus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12x75 *BD Falcon High Clarity Polypropylene Round Bottom Test Tube</td>
			<td style="width:191px;">BD Biosciences</td>
			<td>352063</td>
			<td>FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD 40 um Falcon cell strainers</td>
			<td style="width:191px;">BD Biosciences</td>
			<td>352340</td>
			<td>FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% Trypsin EDTA&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td>25200056</td>
			<td>FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plate, flat bottom, clear&#160;</td>
			<td>Corning&#160;</td>
			<td>3596</td>
			<td>FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well cell culture plates&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS3997</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optilux black clear bottom&#160;</td>
			<td>Corning&#160;</td>
			<td>3603</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flexstation pipet tips</td>
			<td style="width:191px;">Molecular Devices</td>
			<td>9000-0911</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetylcholine Chloride&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A2661</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Norepinephrine&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A7256</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dopamine Hydrochloride&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">PHR1090</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GABA&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A2129</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histamine&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">H7125</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamate&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">49621</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epinephrine&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">E4642</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Somatostatin&#160;&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">S1763</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5HT&#160;&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">H9523</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VIP&#160;</td>
			<td style="width:191px;">Alpha Diagnostics Inc.&#160;&#160;&#160;</td>
			<td style="width:429px;">SP-69627</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orexin A</td>
			<td style="width:191px;">Alpha Diagnostics Inc.&#160;&#160;&#160;</td>
			<td style="width:429px;">12-p-01</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Substance P&#160;&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">S6883</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A4036</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Melatonin&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">M5250C</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Fluorescence Plate Reader &#38; software</td>
			<td style="width:191px;">Molecular Devices</td>
			<td style="width:429px;">Flexstation 3</td>
			<td>Flexstation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;DMEM (high glucose) with Glutamax&#160;&#160;</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">10569-010</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;Fetal bovine serum</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">10082-139</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;Pen/Strep&#160;</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">15140-122</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;Puromycin&#160;&#160;</td>
			<td style="width:191px;">InvivoGen</td>
			<td style="width:429px;">ant-pr-1</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;Fibronectin&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">F0895</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:387px;">CoolCell LX Alcohol-free controlled-rate cell freezing box</td>
			<td style="width:191px;">Bioexpress</td>
			<td style="width:429px;">D-3508)</td>
			<td>Tissue culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">cyanoacrylate glue&#160;</td>
			<td style="width:191px;">Loctite</td>
			<td style="width:429px;">Loctite no. 495</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic paraffin film&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:429px;">Parafilm&#174;</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NANOINJECTOR</td>
			<td style="width:191px;">Drummond</td>
			<td style="width:429px;">3-000-204</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GLASS ELECTRODES</td>
			<td style="width:191px;">Drummond</td>
			<td style="width:429px;">3-000-203G</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hand held drill</td>
			<td style="width:191px;">OSADA</td>
			<td style="width:429px;">Exl-M40</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Burrs for drill</td>
			<td style="width:191px;">Fine Scientific</td>
			<td style="width:429px;">19007-05; 19007-07)</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Sterilizing bath</td>
			<td style="width:191px;">FST</td>
			<td style="width:429px;">18000-45, Hot Bead Sterilizer</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:387px;">isoflurane chamber/mask</td>
			<td style="width:191px;">Highland Medical Equipment</td>
			<td style="width:429px;">564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">3D scope with arm</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">fiber optic light</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Betadine&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70 % (v/v) isopropyl alcohol</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone-Iodine Prep Pads</td>
			<td style="width:191px;">dynarex</td>
			<td style="width:429px;">1108</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl 0.9% (INJECTION, USP, 918610)</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CYCLOSPORINE (INJECTION, USP)</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenex (INJECTION) buprenorphine (0.03 &#956;g per g rodent)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic ointment&#160;</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:429px;">NDC 17478-235-35</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgifoam</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grip dental cement</td>
			<td style="width:191px;">Dentsply</td>
			<td style="width:429px;">#675571, 675572</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instant SuperGlue&#160;</td>
			<td style="width:191px;">NDindustries</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LOCTITE 4041</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">METABOND</td>
			<td style="width:191px;">C&#38;B</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">no. 0 cover glass</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stereotaxic frame&#160;</td>
			<td style="width:191px;">Kopf</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Rectal probe and heating pad</td>
			<td style="width:191px;">FHC</td>
			<td style="width:429px;">40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">optical breadboard for imaging</td>
			<td style="width:191px;">Thorlabs</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mineral oil</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;">S55667</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kwik-Cast (Silicone elastomer)</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suture&#160;&#160;</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;">18&#8217;&#8217;, 1667, 4-0</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91500-09, 15018-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forcepts</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">11252-30; #55, 11295-51; Grafe, 11050-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Student Halsted-Mosquito Hemostats</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91308-12</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small Vessel Cauterizer Kit</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-00</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot Bead Sterilizers</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-45</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instrument Case with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20311-21</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Sterilization Containers with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20810-01</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:387px;">2P fixed-stage fluorescence scope for in vivo imaging</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">FV1200 MPE</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Multiphoton laser</td>
			<td style="width:191px;">SpectraPhysics</td>
			<td style="width:429px;">Mai Tai DeepSee</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Green Laser</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">473 nm Laser</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">xy translation base</td>
			<td style="width:191px;">Scientifica</td>
			<td style="width:429px;">MMBP</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">FRET filter cube for YFP and CFP</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">25-X water immersion objective</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">air table</td>
			<td style="width:191px;">Newport</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">custom built light-tight cage</td>
			<td style="width:191px;">Thorlab</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
	</tbody>
</table>

construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo

세포 기반 신경 전달 형광 설계 기자 (CNiFERs)는 신경 과학자 광학적 생체의 뇌에서 신경 전달 물질의 방출을 검출하기위한 새로운 수단을 제공한다. 특정 CNiFER 안정적 특정 G 단백질 결합 수용체, G의 Q에 커플 / 11 G 단백질 및 FRET 기반 칼슘) 검출기 (, TN-XXL를 표현하는 인간 배아 신장 세포에서 생성됩니다. 수용체의 활성화는 FRET 신호의 증가를 이끈다. CNiFER 클론은 도파민에 대한 D2R, 예를 들어, 특정 신경 전달 물질의 고유 수용체를 이용하기 때문에 CNiFERs는 nM의 감도와 초 시간 응답이 있습니다. CNiFERs 직접 뇌에 이식 생체 내에서 볼륨 전송을 측정하기에 이상적 미만 백 ㎛의 공간 해상도와 신경 전달 물질 방출을 감지 할 수 있도록하고 있습니다. CNiFERs 또한 VI 잠재적 교차 - 반응성에 대한 다른 약물을 스크리닝하는데 사용될 수있다VO. 우리는 최근의 GPCR에게 G I / O G 단백질에 그 부부를 포함 CNiFERs의 제품군을 확장했다. CNiFERs은 아세틸 콜린 (ACH), 도파민 (DA)과 노르 에피네프린 (NE)를 검출 할 수 있습니다. 어떤 GPCR이 CNiFER 소설을 생성하는데 사용될 수 있음을 감안할 때 대략 인간 게놈 800 GPCRs에 있다는 것을 우리는 설계, 실현 및 CNiFER 임의의 타입을 테스트하기 위해 여기 일반적인 방법을 설명한다.

특정 G 단백질 결합 수용체 (GPCR)와 유전자 코드 [칼슘 2 +] 센서, TN-XXL하십시오 CNiFER 안정적 적어도 두 개의 단백질을 발현하도록 유전자 조작 된 인간 배아 신장 (HEK293) 세포에서 파생됩니다. TN-XXL은 칼슘 이온 -6,15-의 질문에 답변 시안과 노란색 형광 단백질 각각 eCFP과 황수정, 사이의 형광 공명 에너지 전달 (FRET)을 겪는다. 내생 G q를 G 단?...

Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

우리는 체적 신경 전달 물질 방출의 광학 검출을위한 셀 기반의 신경 전달 물질의 형광 설계 기자 (CNiFERs)를 만들 수있는 프로토콜을 제시한다.

건설 셀 기반의 신경 전달 물질의 광학 검출을위한 신경 전달 물질 형광 엔지니어링 기자 (CNiFERs)<em&gt; 생체</em

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

Research

JoVE Journal

Neuroscience

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

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

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

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 ...

연구

신경과학

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

에 얇게 - 두개골 두피 창 기술<em&gt; 생체 내</em&gt; 광 결맞음 단층 촬영 영상

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

형광에 의한 신경 조직의 영구 DNA 바이러스의 게놈 및 성적 증명서의 검출 현장에서 하이브리드 면역 염색과 결합

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

모델링 성상 세포종 병인<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 대뇌 피질의 성상이나 조건, 유전자 조작 쥐에서 신경 줄기 세포를 사용하여

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

의 헤어 난포 피침 엔딩에서 생활 신경 터미널 라벨의 광학 모니터링<em&gt; 전의 VIVO</em&gt; 마우스 귀 피부

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

광학 일관성 단층 촬영: 마우스 망막 신경 절 세포에서 Vivo에서 화상 진 찰

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

자연스럽 게 여성 시리아 햄스터에 동작을 보람 있는 하는 동안 Extracellular 신경 전달 물질의 생체 내에서 변화를 측정

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Vivo에서 형광 기자를 사용 하 여 마우스 Suprachiasmatic 핵에서 Circadian 시계 유전자 발현의 모니터링

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...