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

我们感谢B.康克林（美国加州大学旧金山分校），用于提供对G qi5和G QS5的cDNA，A史怀哲寻求帮助，以电子，N.泰勒与克隆，伊恩Glaaser和罗伯特·里夫金的校对筛查援助，和Olivier Griesbeck为TN-XXL。 （; DA037170 DA029706），生物医学成像和生物工程研究所（NIBIB）（EB003832），霍夫曼 - 罗氏（88610A）和“神经科学这项工作是通过药物滥用美国国家研究所（NIDA）的研究资助项目通过NIDA（DA007315）关于滥用“培训资助的药物。

在这项研究中进行的所有动物的程序都是按照机构动物护理和使用委员会（IACUC）的指导方针，并已通过在医学学院伊坎在西奈山和加州大学圣地亚哥分校的IACUCs。 1.生成GPCR表达的慢病毒转化HEK293细胞<ol><li>获得的cDNA为从商业来源， 例如 ，cdna.org特定GPCR。可替代地，扩增从使用PCR的cDNA文库的GPCR基因。获得慢病毒表达载体，如PCDH-CMV-MCS-EF1-迪普罗（PCDH）。使用该向量来传播该DNA以?...

<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>

CNiFERs的创建提供了用于体内光学测量大脑神经递质释放的创新和独特的策略。 CNiFERs非常适合用于测量突触外发布， 即音量传导，神经递质。重要的是，每CNiFER具有天然的GPCR的性质，提供了在大脑中的神经递质的水平的变化的生理光学测量。迄今，已被用于检测乙酰创建CNiFERs（M1-CNiFER）6，多巴胺（D2-CNiFER）7和去甲肾上腺素（α1A-CNiFER）7。 <p class="jove_co...

为了全面了解神经元在大脑中的通信方式，它必须有用于测量神经递质在体内的释放的方法。有在体内神经递质检测几种行之有效的方法。一种常用的技术是微透析，其中套管被插入到脑和脑脊液中的一小体积被收集并用高效液相色谱法和电化学检测1进行分析。微透析有探针的几个直径的量级的空间分辨率， 例如 ，约0.5毫米的直径200微米的微探针。这种技术的时间分辨率，但是，是缓慢由于采样间隔，通常持续约5分钟以上1。此外，分析还没有实时进行。另一种技术是快速扫描循环伏安（FSCV），其使用插入到大脑的碳纤维探针。 FSCV具有优良的温度口服分辨率（亚秒），高灵敏度（毫微摩尔），并与探针直径的5至30微米的空间分辨率。然而，FSCV被限制为在碳势探头2产生的特性的氧化和与电压降低信息发射机。 测量神经递质第三种技术是直接通过基因编码的神经递质（NT）生物传感器3。用这种方法，融合蛋白创建包含用于耦合到荧光共振能量转移（FRET）系一对荧光团4或置换的GFP 5的发射机的配体结合域。不同于前两种方法，这些生物传感器遗传编码和表达的宿主细胞的表面上，如神经元，通过生产转基因动物的或急性与使用病毒试剂的感染细胞。迄今为止，遗传编码的生物传感器已被被仅用于detectin开发摹谷氨酸和GABA 3-5。这些技术的限制一直是低的灵敏度，在nM范围内，以及无法扩大检测到大量的发送器， 例如 ，经典的神经递质，神经肽和神经调质，其通过G蛋白偶联受体（GPCR）的信号。事实上，也有人类基因组近800 G蛋白偶联受体。 为了解决这些不足，我们已经开发了一种创新工具，通过一个GPCR信号的任何神经递质的光学测量释放。 CNiFERs（基于细胞的神经递质的荧光工程记者）的设计，以表达特定的GPCR是，在受到刺激时，会触发钙离子浓度由遗传编码的基于FRET的钙离子传感器检测到细胞内增加克隆HEK293细胞， TN-XXL。因此，CNiFERs变换神经递质受体结合到荧光变化，提供了一个直接和实时的光读EAD出局部神经递质的活动。通过利用天然受体对于给定的神经递质，CNiFERs保留化学特异性，亲和力和内源性表达的受体的时空动力 ​​学。到目前为止，我们已经创建了三种CNiFERs，一个用于使用M1受体，一个用于检测多巴胺检测乙酰胆碱使用D2受体，和一个用于使用α1A受体6,7-检测去甲肾上腺素。该CNiFER技术很容易扩展和可扩展性，使得它适合于任何类型的GPCR的。在这个朱庇特的文章中，我们描述和示出了在体内 CNiFERs设计，实现的方法，及测试方法的任何应用。

<table border="0" cellpadding="0" cellspacing="0" width="1212">
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="20" style="height:20px;">Adenosine</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A4036</td>
			<td>Flexstation</td>
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			<td height="20" style="height:20px;">Melatonin&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">M5250C</td>
			<td>Flexstation</td>
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			<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>
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			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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			<td height="20" style="height:20px;width:387px;"></td>
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			<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

基于细胞的神经递质荧光工程记者（CNiFERs）为神经科学家光学检测神经递质的释放在脑中体内的新工具。具体CNiFER是从稳定地表达特定的G蛋白偶联受体，其耦合至G Q / 11的G蛋白，以及一个基于FRET的钙 -detector，TN-XXL人胚肾细胞产生。该受体的活化导致的增加的FRET信号。 CNiFERs有纳米灵敏度和几秒钟的时间响应，因为一个CNiFER克隆利用天然受体对特定的神经递质， 例如 D2R为多巴胺。 CNiFERs被直接注入到大脑，使他们能够感神经递质释放与小于一百微米的空间分辨率，这使得它们理想来测量体内体积传输。 CNiFERs也可以用来筛选其它药物用于在vi潜在的交叉反应性VO。我们最近扩大CNiFERs的系列，包括G蛋白偶联受体的夫妇至G I / O的G蛋白。 CNiFERs可用于检测乙酰胆碱（ACh），多巴胺（DA）和去甲肾上腺素（NE）。鉴于任何GPCR可用于创建一个新颖CNiFER并有大约在人类基因组800的GPCR，我们在这里描述的一般方法来设计，实现和测试的任何类型的CNiFER的。

特定G蛋白偶联受体（GPCR）和遗传编码的[Ca 2+]传感器，TN-XXL：一个CNiFER是从被设计为稳定地表达至少两种蛋白质人胚肾（HEK293）细胞衍生。 TN-XXL经受响应青色和黄色荧光蛋白，分别ECFP和黄晶，之间荧光共振能量转移（FRET）来的Ca 2+离子6,15。的GPCR即耦合到内源性ģq的G蛋白的激活触发胞质增加的[Ca 2+]通过PLC / IP 3通路，导致...

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

我们提出了一个协议，用于容量神经递质释放的光学检测创建基于细胞的神经递质荧光工程记者（CNiFERs）。

构建基于细胞的神经递质荧光工程记者（CNiFERs）神经递质的光学检测<em&gt;在体内</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

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

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

科研

神经科学

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

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

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

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

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

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

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

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

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

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

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

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

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

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

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

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

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

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

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