Name: quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo
Uploaded: 2019-06-28T14:45:00
Description: Watch this Scientific Journal Video about Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo at JoVE.com

作者感谢曾燕夏对小鼠的基因分型,汤姆·伯林的氨基酸和薄膜的加工,和梅琴进行一些rCPS实验。这项研究得到了NIMH的内学研究计划,ZIA MH00889的支持。RMS 还得到了自闭症讲博士后奖学金 8679 和 FRAXA 博士后奖学金的支持。

注:所有动物程序均获得国家精神卫生动物护理和使用委员会的批准,并按照国家卫生研究院《动物护理和使用指南》进行。
图 1中提供了该协议的概述。
1. 分别在股骨静脉和动脉中植入导管,分别用于给导管和采集时导动脉血样。在给动剂前至少22小时完成手术。手术大约需要1小时才能完成。
<ol><li>收集必要的材料:无菌手术器械(手...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+local+cerebral+protein+synthesis+in+fetal+and+neonatal+sheep.">Rates of local cerebral protein synthesis in fetal and neonatal sheep.</a> The American Journal of Physiology. 272, R1235-R1244 (1997).</li><li>Nakanishi, H., 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=Positive+correlations+between+cerebral+protein+synthesis+rates+and+deep+sleep+in+Macaca+mulatta.">Positive correlations between cerebral protein synthesis rates and deep sleep in Macaca mulatta.</a> The European Journal of Neuroscience. 9, 271-279 (1997).</li><li>Sun, Y., Deibler, G. E., Sokoloff, L., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+regional+rates+of+cerebral+protein+synthesis+adjusted+for+regional+differences+in+recycling+of+leucine+derived+from+protein+degradation+into+the+precursor+pool+in+conscious+adult+rats.">Determination of regional rates of cerebral protein synthesis adjusted for regional differences in recycling of leucine derived from protein degradation into the precursor pool in conscious adult rats.</a> Journal of Neurochemistry. 59, 863-873 (1992).</li><li>Scammell, T. E., Schwartz, W. J., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+a+circadian+rhythm+of+protein+synthesis+in+the+rat+suprachiasmatic+nuclei.">No evidence for a circadian rhythm of protein synthesis in the rat suprachiasmatic nuclei.</a> Brain Research. 494, 155-158 (1989).</li><li>Smith, C. B., Eintrei, C., Kang, J., Sun, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+thiopental+anesthesia+on+local+rates+of+cerebral+protein+synthesis+in+rats.">Effects of thiopental anesthesia on local rates of cerebral protein synthesis in rats.</a> The American Journal of Physiology. 274, E852-E859 (1998).</li><li>Sun, Y., Deibler, G. E., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+axotomy+on+protein+synthesis+in+the+rat+hypoglossal+nucleus:+examination+of+the+influence+of+local+recycling+of+leucine+derived+from+protein+degradation+into+the+precursor+pool.">Effects of axotomy on protein synthesis in the rat hypoglossal nucleus: examination of the influence of local recycling of leucine derived from protein degradation into the precursor pool.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 13, 1006-1012 (1993).</li><li>Smith, C. B., Yu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+protein+synthesis+in+the+regenerating+hypoglossal+nucleus:+effects+of+testosterone+treatment.">Rates of protein synthesis in the regenerating hypoglossal nucleus: effects of testosterone treatment.</a> Neurochemical Research. 19, 623-629 (1994).</li><li>Orzi, F., Sun, Y., Pettigrew, K., Sokoloff, L., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+and+delayed+effects+of+prior+chronic+cocaine+administration+on+regional+rates+of+cerebral+protein+synthesis+in+rats.">Effects of acute and delayed effects of prior chronic cocaine administration on regional rates of cerebral protein synthesis in rats.</a> The Journal of Pharmacology and Experimental Therapeutics. 272, 892-900 (1995).</li><li>Nadel, 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=Voluntary+exercise+regionally+augments+rates+of+cerebral+protein+synthesis.">Voluntary exercise regionally augments rates of cerebral protein synthesis.</a> Brain Research. 1537, 125-131 (2013).</li><li>Sun, 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=Rates+of+local+cerebral+protein+synthesis+in+the+rat+during+normal+postnatal+development.">Rates of local cerebral protein synthesis in the rat during normal postnatal development.</a> The American Journal of Physiology. 268, R549-R561 (1995).</li><li>Smith, C. B., Sun, Y., Sokoloff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+on+regional+rates+of+cerebral+protein+synthesis+in+the+Sprague-Dawley+rat:+examination+of+the+influence+of+recycling+of+amino+acids+derived+from+protein+degradation+into+the+precursor+pool.">Effects of aging on regional rates of cerebral protein synthesis in the Sprague-Dawley rat: examination of the influence of recycling of amino acids derived from protein degradation into the precursor pool.</a> Neurochemistry International. 27, 407-416 (1995).</li><li>Ingvar, M. C., Maeder, P., Sokoloff, L., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aging+on+local+rates+of+cerebral+protein+synthesis+in+rats.">The effects of aging on local rates of cerebral protein synthesis in rats.</a> Monographs in Neural Sciences. 11, 47-50 (1984).</li><li>Sare, R. M., Huang, T., Burlin, T., Loutaev, I., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+rates+of+cerebral+protein+synthesis+measured+in+vivo+in+a+mouse+model+of+Tuberous+Sclerosis+Complex:+unexpected+consequences+of+reduced+tuberin.">Decreased rates of cerebral protein synthesis measured in vivo in a mouse model of Tuberous Sclerosis Complex: unexpected consequences of reduced tuberin.</a> Journal of Neurochemistry. 145, 417-425 (2018).</li><li>Liu, Z. H., Huang, T., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium+reverses+increased+rates+of+cerebral+protein+synthesis+in+a+mouse+model+of+fragile+X+syndrome.">Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome.</a> Neurobiology of Disease. 45, 1145-1152 (2012).</li><li>Qin, M., 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=Altered+cerebral+protein+synthesis+in+fragile+X+syndrome:+studies+in+human+subjects+and+knockout+mice.">Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 33, 499-507 (2013).</li><li>Qin, M., Kang, J., Burlin, T. V., Jiang, C., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postadolescent+changes+in+regional+cerebral+protein+synthesis:+an+in+vivo+study+in+the+FMR1+null+mouse.">Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse.</a> The Journal of Neuroscience: the Official Journal of the Society for Neuroscience. 25, 5087-5095 (2005).</li><li>Qin, M., 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=R-Baclofen+Reverses+a+Social+Behavior+Deficit+and+Elevated+Protein+Synthesis+in+a+Mouse+Model+of+Fragile+X+Syndrome.">R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome.</a> The International Journal of Neuropsychopharmacology. 18, pyv034 (2015).</li><li>Qin, M., 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=Cerebral+protein+synthesis+in+a+knockin+mouse+model+of+the+fragile+X+premutation.">Cerebral protein synthesis in a knockin mouse model of the fragile X premutation.</a> ASN Neuro. 6, (2014).</li><li>Smith, C. 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提出了一种定量测定实验动物体内脑蛋白合成(rCPS)区域速率的方法。与现有方法的方法,这种方法具有相当大的优势:1.测量是在清醒的有行为动物中进行的,因此它们反映了大脑中正在进行的过程。2. 测量是通过定量自辐射法进行的,能够同时确定大脑所有区域和分区域的rCPS。3. 该方法的动力学模型考虑到从组织蛋白降解中产生的未标记氨基酸的回收可能性及其对蛋白质合成前体池的影响6。<sup class=...

蛋白质合成是神经系统长期适应性变化的重要生物过程。抑制蛋白质合成会阻止无脊椎动物和脊椎动物的长期记忆储存2。蛋白质合成对于维持某些形式的长期强效 (LTP) 和长期抑郁症 (LTD) 3、发育过程中的神经元存活率4以及神经元及其一般维持的后期阶段至关重要突触连接5.大脑蛋白质合成率的测量可能是研究适应性变化以及与学习和记忆相关的神经发育障碍和紊乱的重要工具。
我们已经开发出一种量化清醒动物体内脑蛋白合成率的方法,它与其他估计脑组织外体或体外制剂速率的技术具有固有的优势。最重要的是适用于清醒动物大脑中完整大脑的测量。这是一个关键的考虑因素,因为它允许具有突触结构和功能的测量,而不必担心验尸后的效果。此外,我们采用的定量自动放射学方法实现了高度的空间定位。虽然14C的能量是,我们不能本地化示踪剂在亚细胞或细胞水平,我们可以测量速率的细胞层和小大脑区域,如下丘脑核,大约25μm分辨率7。
用放射性追踪器进行体内测量的一个挑战是确保测量的放射性标签是在感兴趣的反应产物中,而不是未反应的标记前体或其他无关标记的代谢产物6。我们选择L-1-14 C+-亮氨酸作为示踪氨基酸,因为它要么被整合到蛋白质中,要么迅速代谢成14CO2,在大脑中未标记的CO2大池中稀释,因为高速率能量代谢8.此外,任何14C未纳入蛋白质主要存在作为自由[14 C]-亮氨酸,在60分钟的实验期间,几乎完全从组织6清除。蛋白质然后固定到组织与形式素,然后用水冲洗,以去除任何免费的 [14C]-亮氨酸之前,自体。
另一个重要考虑因素是组织蛋白解中未标记氨基酸稀释前体氨基酸池特定活性的问题。我们已经表明,在成年大鼠和小鼠中,大脑中蛋白质合成的前体亮氨酸池中约有40%来自来自蛋白质分解6的氨基酸。这必须包括在脑蛋白合成(rCPS)的区域速率的计算中,并且必须在可能改变这种关系的研究中得到确认。该方法的理论基础和假设已在其他地方详细介绍。本文着重论述了该方法应用的程序问题。
该方法已用于测定地松鼠9,绵羊10,河河猴11,大鼠12,13,14,15,16的rCPS,17,18,19,20,21、一种小鼠模型的管状硬化复合22,小鼠模型脆弱的X综合征23,24,25,26,脆弱的X预突变小鼠27,和苯丙酮尿的小鼠模型28。在本手稿中,我们介绍了使用体内自放射学 L-1-14 C+-亮氨酸法测量rCPS 的程序。我们在醒着的控制鼠标的大脑区域中呈现rCPS。我们还证明,在体内对一种翻译抑制剂的抗声霉素的分管,可以消除大脑中的蛋白质合成。

<table>
	<tbody>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>003024</td>
			<td>Fmr1 knockout breeding pairs</td>
		</tr>
		<tr>
			<td>Anisomycin</td>
			<td>Tocris Bioscience</td>
			<td>1290</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematocrit Tubes</td>
			<td>Drummond Scientific</td>
			<td>1-000-3200-H</td>
			<td>capillary tubes</td>
		</tr>
		<tr>
			<td>Critoseal Capillary Tube Sealant</td>
			<td>Leica Microsystems</td>
			<td>39215003</td>
			<td>sealant putty</td>
		</tr>
		<tr>
			<td>Glass vial inserts</td>
			<td>Agilent</td>
			<td>5183-2089</td>
			<td>used to collect blood samples</td>
		</tr>
		<tr>
			<td>Digi-Med Blood Pressure Analyzer</td>
			<td>Micro-Med Inc.</td>
			<td>BPA-400</td>
			<td>blood pressure analyzer</td>
		</tr>
		<tr>
			<td>Bayer Breeze 2 Blood Glucose Monitoring System</td>
			<td>Bayer Breeze</td>
			<td>9570A</td>
			<td>glucose meter</td>
		</tr>
		<tr>
			<td>Gastight syringe</td>
			<td>Hamilton Co.</td>
			<td>1710</td>
			<td>tuberculin glass syringe</td>
		</tr>
		<tr>
			<td>HeatMax HotHands-2 Hand Warmers</td>
			<td>HeatMax</td>
			<td>Model HH2</td>
			<td>warming pads</td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution</td>
			<td>Fresenius Kabi USA, LLC</td>
			<td>504505</td>
			<td>heparin saline</td>
		</tr>
		<tr>
			<td>Clear animal container</td>
			<td>Instech</td>
			<td>MTANK/W</td>
			<td>animal enclosure</td>
		</tr>
		<tr>
			<td>Spring tether</td>
			<td>Instech</td>
			<td>PS62</td>
			<td>catheter tube/rodent attachment</td>
		</tr>
		<tr>
			<td>Swivel</td>
			<td>Instech</td>
			<td>375/25</td>
			<td>hooks to spring tether</td>
		</tr>
		<tr>
			<td>Swivel arm and mount</td>
			<td>Instech</td>
			<td>SMCLA</td>
			<td>hooks to swivel and animal enclosure</td>
		</tr>
		<tr>
			<td>Tether button</td>
			<td>Instech</td>
			<td>VAB62BS/22</td>
			<td>attaches to bottom of spring tether</td>
		</tr>
		<tr>
			<td>Stainless steel tube</td>
			<td>Made in-house</td>
			<td>N/A</td>
			<td>used to snake catheters through mouse</td>
		</tr>
		<tr>
			<td>Matrx VIP 3000</td>
			<td>Matrx</td>
			<td>91305430</td>
			<td>isoflurane vaporizer</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Stoelting Co.</td>
			<td>50207</td>
			<td>isoflurane/halothane adsorber</td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>Oster Finisher</td>
			<td>Model 59</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical skin hooks</td>
			<td>Made in-house (??)</td>
			<td>N/A (??)</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride Saline</td>
			<td>APP Pharmaceuticals LLC</td>
			<td>918610</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>UNIFY silk surgical sutures</td>
			<td>AD Surgical</td>
			<td>#S-S618R13</td>
			<td>6-0 USP, non-absorbable</td>
		</tr>
		<tr>
			<td>PE-8 polyethylene tubing</td>
			<td>SAI Infusion Technologies</td>
			<td>PE-8-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton Dickinson and Co.</td>
			<td>309659</td>
			<td>1cc/mL</td>
		</tr>
		<tr>
			<td>PE-10 polyethylene tubing</td>
			<td>Clay Adams</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr>
			<td>MCID Analysis</td>
			<td>Imaging Research Inc.</td>
			<td>Version 7.0</td>
			<td>optical density analysis</td>
		</tr>
		<tr>
			<td>Gelatin-coated slides (75x25mm)</td>
			<td>FD Neurotechnologies</td>
			<td>PO101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Super RX-N medical x-ray film</td>
			<td>Fuji</td>
			<td>47410-19291</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypercassettes (8x10 in)</td>
			<td>Amersham Pharmacia Biotech</td>
			<td>11649</td>
			<td></td>
		</tr>
		<tr>
			<td>[1-14C]leucine</td>
			<td>Moravek</td>
			<td>MC404E</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Sarstedt Aktiengesellschaft &#38; Co.</td>
			<td>72.692.005</td>
			<td>used to deproteinize blood samples</td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>Wheaton</td>
			<td>357335</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>Sigma-Aldrich</td>
			<td>18421</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>NIH Supply Center</td>
			<td>6830009737285</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation fluid</td>
			<td>CytoScint</td>
			<td>882453</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintilllation counter</td>
			<td>Packard Tri-Carb</td>
			<td>2250CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino acid analyzer</td>
			<td>Pickering Laboratories</td>
			<td>Pinnacle PCX</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC unit</td>
			<td>Agilent Technologies</td>
			<td>1260 Infinity</td>
			<td>include 1260 Bio-Inert Pump</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>M650</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfosalicylic acid</td>
			<td>Sigma-Aldrich</td>
			<td>MKBS1634V</td>
			<td>5-sulfosalicylic acid dihydrate</td>
		</tr>
		<tr>
			<td>Norleucine</td>
			<td>Sigma</td>
			<td>N8513</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 N HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>[H3]leucine</td>
			<td>Moraevk</td>
			<td>MC672</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tube</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>50 mL conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Heuer Microsplit</td>
			<td>Model 1000</td>
			<td>1/100 min</td>
		</tr>
		<tr>
			<td>Euthanasia Solution</td>
			<td>Vet One</td>
			<td>H6438</td>
			<td></td>
		</tr>
		<tr>
			<td>Northern Light Precision Illuminator</td>
			<td>Imaging Research Inc.</td>
			<td>Model B95</td>
			<td>fluorescent light box</td>
		</tr>
		<tr>
			<td>Micro-NIKKOR 55mm f/2.8</td>
			<td>Nikon</td>
			<td>1442</td>
			<td>CDD camera</td>
		</tr>
	</tbody>
</table>

quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo

蛋白质合成是神经元功能发育和维护所必需的,并参与神经系统的适应性变化。此外,人们认为神经系统蛋白质合成障碍可能是某些发育障碍的核心表型。准确测量动物模型中脑蛋白合成率对于了解这些疾病非常重要。我们开发的方法被设计用于研究醒着、有行为的动物。它是一种定量的自动放射学方法,因此可以同时产生大脑所有区域的速率。该方法基于示踪氨基酸L-1-14 C+-亮氨酸的使用,以及L-亮氨酸在大脑中行为的动力学模型。我们选择L-1-14 C+-亮氨酸作为示踪剂,因为它不会导致外在标记的代谢产物。它要么被整合到蛋白质中,要么被快速代谢,产生14CO2,在大脑中大量未标记的CO2中稀释。 该方法和模型还允许从组织蛋白解基中提取的无标记亮氨酸对蛋白质合成的组织前体池的贡献。该方法具有空间分辨率,可确定细胞和神经层以及下丘脑和颅神经核的蛋白质合成率。为了获得可靠且可重复的定量数据,必须遵循程序细节。这里我们介绍了定量自放射学L-1-14 C+-亮氨酸法的详细程序,用于确定体内蛋白质合成的区域速率。

这里展示了一个代表性的实验,演示了蛋白质合成抑制剂先前对rCPS的影响。正常盐水中的苯二恶客在开始rCPS测定前30分钟给一只成年C57/BL6雄性野生型小鼠皮下(100毫克/千克)施用。与车辆处理的对照动物相比,异种素治疗的效果表明,在异种霉素处理小鼠中,rCPS几乎无法检测到(图4)。这些数据表明,体内自放射学L-1-14 C+-亮氨酸法测量大脑中蛋白质?...

Watch this Scientific Journal Video about Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo at JoVE.com

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

蛋白质合成是细胞的关键生物过程。在大脑中,它是自适应变化所必需的。测量完整大脑中蛋白质合成率需要仔细的方法学考虑。这里我们介绍了L-[1-14C]-亮细胞定量自射方法,用于测定体内脑蛋白合成的区域速率。

定量自动放射法测定维沃脑蛋白合成区域速率

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

测量脑蛋白合成的区域速率可以追踪大脑对发育和神经可塑性期间发生的长期变化的反应。我们的方法的优点是测量是完全定量的，他们是在清醒的行为动物。定量自动放射技术允许同时在所有大脑区域进行测量。演示这个程序的将是我实验室的博士后研究员安妮塔·托罗西安和我们的动物外科医生黄天健。从准备手术开始，如文本协议中详述。一旦进入手术阶段，使用手术剪刀从左大腿上部中侧向中线切口切口一厘米，露出股动脉和静脉。用手术皮肤钩在切口的上方和两侧收回松弛的皮肤。通过将皮钩贴在手术阶段，固定它们。将无菌 0.9% 氯化钠涂抹到暴露区域，以保持足够的水分。使用钳子钝解剖，分离股动脉和静脉的一小部分周围的结缔组织。小心地分离动脉和静脉。现在使用钳子在切口的最横向点将一股可吸收缝合线串在股骨静脉和动脉下。将缝合线拉过一半，使末端均匀。在腹股沟的更近点，使用钳子在股骨静脉下螺纹第二个缝合线。轻轻系上半结，用来限制血流。在股A和股B之间的一个点，使用钳子在股骨静脉下螺纹第三个缝合线。轻轻打一个用于限制血液流动的满满结。小心不要撕裂静脉。轻轻拖曳 B 链以限制血液流动。使用血缘轻轻拖曳链 B 以保持血液限制。现在，将 PE 管的非切端连接到 32 量表针和充满肝素盐水的 1 毫米注射器。冲洗导管以清除气泡。用微型剪刀在股骨静脉的禁区内切一个小孔，并小心地将冲洗的PE八管的角端插入到股B上。一旦插入，释放股B的张力，引导导管进一步向上静脉。在包含导管的静脉周围拧紧链 B。使用链 C，在导管周围再系一个结。确保这个结不会捕获股动脉。轻轻拉回注射器筒，以部分填充带血，以确保导管已正确植入，然后使用相同的程序将 PE 10 导管插入左股骨动脉。股骨静脉和动脉导管都固定住后，将链A系成两个导管周围的结。切除所有多余的缝合线并去除皮钩后，用肝素盐水冲洗动脉导管，以防止凝血。将两个导管的末端烧化以创建密封件。将鼠标放在易发位置，并在颈部底部进行一个小切口，将盐水涂在暴露区域。将空心金属棒从颈部切口潜到股骨切口。蛇导管通过空心棒和颈部切口。取出空心棒后，用缝合关闭股骨切口，然后进行手术后镇痛。蛇导管通过一个30厘米的柔性空心管，使弹簧系绳，然后将弹簧系绳的按钮放在皮肤下，然后是手术后镇痛。在恢复期间，将鼠标移到一个清晰的圆柱形容器中，并配有旋转支座和手臂来装住鼠标。在容器下面放置一个暖手器，以保持鼠标温暖。通过采集文本协议中详细显示的样本，确保小鼠在实验开始时处于正常生理状态。要静脉内施用示踪剂，请使用带注射器的 Y 连接器，注射器将 C 14 标有亮氨酸示踪剂连接到一只手臂，使用带 100 至 200 微升无菌盐水的注射器，以冲洗连接到另一只手臂的静脉线。将 Y 连接器连接到静脉线。启动研究，同时启动停止观察，注射示踪剂，并收集及时动脉血样。注射后立即用盐水冲洗静脉线。在实验前两分钟，以同样的方式连续采集一至七个血样。收集7个样本后，在剩余的样本之前收集30微升的死空间血。样本8至14分别采集3、5、10、15、30、45和60分钟。在实验过程中，在实验过程中，处理三根管的内部标准，其中含有三分开的白血病和诺列素，详见文本协议。要量化血浆白化素浓度，请使用带钠阳离子交换柱的 HPLC 系统，使用带正面甲醛和面粉测量检测的柱后派生。列氨酸曲线下的区域与样品中列氨酸的浓度成正比。使用与标准的比较来量化样品中的列氨酸浓度。然后使用液体闪烁计数器来量化血浆样品中每分钟的三氧化钛和 C 14 的分解。使用这些浓度从循环及其在动脉等离子体中特定活动的时间过程构造C14标记的白化素的间隙曲线。从该图中计算动脉等离子体中标有白化特异性的集成 C 14。要进行定量自皮造影，请准备厚度为20微米的脑科。在零下20摄氏度的低温下通过低温恒温器切分大脑。明胶涂层幻灯片上的解冻安装部分。固定幻灯片后，将幻灯片与一组以前校准的 C 14 标记的甲基丙烯酸酯标准一起安排在 X 射线胶片盒中。在黑暗的房间里，在安全的灯光下，放置一块X射线薄膜，乳液侧向下的两侧和标准。密封盒式磁带，并放在黑色更换袋中。根据制造商的说明开发薄膜。不建议自动薄膜开发，因为背景可能不均匀，并且会影响定量。根据薄膜上一组校准标准的光学密度值，构建光学密度与组织 C 14 浓度的校准曲线。将这些数据拟合于多项式。第二度或第三度多项式方程非常合适。要分析特定的大脑区域，请通过与大脑图集进行比较，将感兴趣区域或 ROI 定位在六到八个部分。记录所有部分中 ROI 中像素的光学密度。根据校准曲线，计算每个像素中的组织 C 14 浓度。最后，从平均组织C14浓度中计算脑蛋白合成的区域速率，在未标记和标记的白化素在t和兰巴的动脉血浆浓度积分中，在ROI中计算。来自血浆的组织前体池中列氨酸的分数。这里展示的具有代表性的图像来自车辆处理的动物相比，用食isoncin治疗的动物，蛋白质合成的抑制剂。蛋白质合成速率与图像中的黑暗程度成正比。阿尼索霉素大幅降低脑蛋白合成的测量速率，表明这种方法的特异性。在这里，数字化的自动放射图显示在海马和下丘脑水平的清醒行为鼠标。较暗区域脑蛋白合成的区域率较高。此处显示的是一个数字化的自动放射图，来自一个清醒的行为控制鼠标在后海马的水平上。根据色条，脑蛋白合成速率在图像中涂上颜色。在尝试此程序时，请务必确保动物在测量过程中处于正常的生理状态。我们的方法已经证明，在神经发育障碍，如脆弱X综合征，蛋白质合成的不管制。它也可能是退行性变化和条件，如阿尔茨海默病的有用标记。蛋白质合成方法可与交替部分的免疫组织化学结合使用，将蛋白质合成的变化与特定蛋白质的区域变化相关。综上关于定量自放射图L-1 C 14白化素法的精确测定，是准确测定体内蛋白质合成区域速率的的理想方法。它在准确性和对体内条件的适用性方面具有相当大的优势。

Research

JoVE Journal

Neuroscience

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...