The authors have nothing to disclose.

所有的程序和行为测试正在协议号12年6月11日，这是符合加拿大理事会的管理和使用实验动物的动物护理的指导方针批准MacEwan大学的动物研究伦理委员会。 1.准备配料罐，解决方案和管理计划<ol><li>得到给药时间表，使得给予动物在相同的环境，并在一天的同一时间，以避免时间或偏压任何的困惑视觉刺激。 </li><li>获得尽可能多的相同的，1.5L的，清晰的聚丙烯产卵所必需的组尺寸的数量的坦克。使用基团的每罐8鱼，其允许2组鱼的要每天稍后在过程中进行测试（参见步骤3）。使用1保持槽和每组一个配料罐（组2×总数）。 <ol><li>将400微米产卵插入所有保温水箱。 ˚F患病与生境的水或反渗透水在适当的温度坦克（斑马鱼，25-28°C），它与温度一致鱼通常安置在。 注意：可以有一些药物和缓冲栖息地水的化学组分之间不希望的化学相互作用。在这种情况下，使用缓冲的最小或没有水族馆的盐用于药物给药反渗透水，以及用于对照组。 </li><li>确保坦克处于中立的环境，以避免给药过程中调节鱼的外部视觉刺激。 </li></ol></li><li>制备药液。混在产卵箱的药物与人居水适量。通过组合3毫升的高品位的乙醇（95％非变性乙醇）与1497毫升的水制备的0.2％的乙醇溶液。通过组合21毫升乙醇与1479毫升的水制备的1.4％的乙醇溶液。 </li></ol> 2.组网鱼和EThanol行政程序<ol><li>从它们的栖息地仔细坦克净鱼转移到含有产卵插入适当的储存罐。理想的情况是，房子在插入产卵鱼，以消除扣除干脆。 </li><li>所有的鱼在各自的储水槽，轻轻抬起产卵插入了贮水箱，并将其放入相应的药液罐（ 图1A）。 <ol><li>根据需要记录的给药时间。用30分钟，在此处描述的过程的乙醇溶液。 </li><li>如果可能的话，有助理协助各实验组的传输同步进行的药液，以确保精确的计量时间。另外，转移一组的时间和跟踪个别群体的计量时间（ 图1A）。 </li></ol></li><li>在必要的给药期结束时，从乙醇溶液小心地提起产卵刀片去掉鱼出的药液，并把轻轻回贮水箱。 </li><li>轻轻地网鱼在暂养箱，把它们放回它们的栖息地，直到下一个预定的时间给药，或将产卵插回储存罐，消除网。 </li><li>作为动物壳体设备，房屋，用作贮水箱在同一水箱和产卵插入动物的参数范围内以前，如果可能的提及。这将消除管理过程中完全扣除。 </li></ol> 3.行为测试<ol><li> 55厘米长9.5厘米深的有白色防水地板（ 图1B）获得光/暗的舞台宽9.5厘米。贴上白色和黑色防水不反光的纸张使用魔术贴竞技场的内壁，其中一半盖在白色的半盖在黑色的舞台上。填写竞技场5厘米的栖息地水的深度在25-28℃的温度。 MAINT艾因在整个测试这个温度。 </li><li>通过构造一个白色的三面外壳为竞技场可以位于Minimalize外部视觉刺激。确保在测试区域具有扩散顶灯灯光不会导致在水面上的反射，但是足够亮的运动跟踪软件，或交-hoc手册量化从视频图像。 </li><li>将舞台上的外壳，并设置行为跟踪软件的记录和运动分析参数。将测试时间为5-15分钟，这取决于所研究的问题。 注意：在这里，我们用了5分钟。 </li><li>运输组鱼进行测试，以研究区的生境坦克，并放置在舞台上外壳的外面。适应的鱼10分钟。 </li><li>轻轻净一个从在光/暗竞技场的中心相应的组和地方鱼，一定要松开鱼，当它被定位成平行于所述舞台的长轴AVOID偏置鱼光或暗区。 </li><li>开始的动物被释放后立即记录的行为。观看任何软件问题跟踪鱼或鱼跳跃或冻结。旋转舞台上的180°后一半的受试者进行了测试，以防止任何的困惑，由于导致从竞技场的一端朝向机箱的开口端的偏见。 </li><li>经过庭审已经结束了，轻轻地网，从舞台上拆下来的鱼保持罐或栖息地的坦克。 </li></ol> 4.分析<ol><li>检查时间花在光与暗区。对于每个组，每个鱼，得到在光与暗区所花费的相对时间，并使用一个样本t检验进行分析（;从总试验时间（分）150秒差或Wilcoxon符号秩检验对非参数数据）的以确定群体显著喜欢一个区域比其他。 </li><li>为了比较偏好，计算prefe减去花费在从在黑暗区中花费的时间的光带和比较各组之间的差异的时间伦斯索引。 叔测试可以用来比较两个组。比较多组同方差的利用Tukey的HSD post hoc检验的单向分析在必要时（或Kruskal-Wallis检验与唐恩的多重比较事后检验用于非参数数据）。 </li><li>比较速度，区转换，曲折的数目，以及各组不动。使用方差利用Tukey的HSD post hoc检验单向分析在必要时（或Kruskal-Wallis检验与唐恩的多重比较事后检验用于非参数数据）。 </li></ol>

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作者承认约书亚盖洛普对使用其用于图1的拍摄设备。这项工作是由自然科学和工程研究理事会（NSERC），加拿大发现金（以TJH）的支持。

涉及药品监督管理局在斑马鱼以往的研究仅仅依靠扣除鱼从家里罐运送他们向药液12,16。网并不总是一致的，往往需要较长的时间比由于斑马鱼，其具有显著个体差异的逃避反应预期。传统的转印方法虽然有用，但也能够在通过减少总时间的鱼花的水以外的量，以及减少在动物之间的传输时间的变化量的改善。除了提高实用性，该方法为研究人员剂量大组鱼的同时在对于时间的精确量的解...

斑马鱼（ 斑马鱼）是一个小的硬骨鱼类物种原产于印度是一个有用的模式生物行为1和医学研究2,3。斑马鱼也常用于各种药理物质的检测，以表征其对行为的影响。不同剂量和给药时间表已经被用于研究斑马鱼的，如兴奋剂4，抗焦虑药5和乙醇6-8化合物给药后的行为。 我们的实验室已调查的充分验证的光/暗检测9,20，通常也被称为scototaxic测定不同乙醇给药时间表对斑马鱼焦虑和运动的影响。开发乙醇给药的新方法，以提高效率为重复，日常管理过的很长一段时间（21天）6 </sUP&gt;。以前使用的方法是实用的，但是，我们试图开发一种方法，该方法减少了网，而其相关联的时间成本，并允许感兴趣的大量鱼的药物的同时，精确地定时给药。在传统的使用研究乙醇中，斑马鱼网状，并从一个罐转移到另一个含有乙醇和水10-12相应的混合物。虽然这种方法是被广泛接受的，扣除斑马鱼可能增加的可变性中引入和从药液去除鱼所需要的时间。因此，精确的曝光于所关注的化合物可能会发生变化对涉及重复给药的实验过程。一种方法是减少错误的变性运输时间所产生的来源是这样希望的。用我们的方法，我们能够同时移动所有的鱼，导致鱼的每一个相同的给药时间。以下乙醇曝光（此处描述的），斑马鱼可以在任何numbe被测试- [R行为测定法，包括那些评估焦虑。使用新的方法给药鱼群体已经超越精确复制和学科之间和整个鱼类群体规范计量能力的实际用途。新软件的出现，允许多个鱼的跟踪立刻可以看到研究人员利用我们的方法，以确保在他们的实验中可复制性和准确性。考虑到广泛使用斑马鱼作为模式生物进行行为神经科学，这种方法会提高效率和实用性于未来的药理研究。 在本范例中，重复给药方案被采用，大约有反映人类饮用的时间表。鱼被随机分成3组：对照组，每天中度或每周-狂欢。给药方案是在持续21天，选择，因为它显著在以前的研究中超过7曝光时间。控制鱼获得零ALCOHOL，每天中度鱼获得0.2％的酒精每天一次，以及每周 - 狂欢的鱼，每周收到1.4％的酒精一次。亮/暗任务，使用后第2天停药的评估焦虑。这是一个相对简单的测试来管理其使用的矩形舞台，其中在一侧的壁是白色并且在另一侧是暗9。成年斑马鱼更喜欢稳健的控制条件6,9,13下舞台的黑暗的一面。增加焦虑操作性定义为显著更多的时间在黑暗中度过区，降低焦虑可以当鱼花比较多的时间在光区花假设。随着运动追踪软件，其他的信息变量也可以量化，包括平均速度，不动，蜿蜒和过渡区的14。 在我们实验室开发的给药方法可以应用到其中的水溶性化合物被施用到一个或多个zebrafi任何研究SH。许多其他药剂可从该方法中受益，目前正在斑马鱼试验。常用的测试化合物包括尼古丁，利眠宁，丁螺环酮，和东莨菪碱，它们溶解在一种类似的方式，以乙醇;通过混合化学适量的水中。因此，该过程的一般范围更广，而不是仅限于乙醇。另外，用药物为多天的给药后，光/暗任务只有一个的许多行为测试，可以被采用。给药后或抽出时，可使用的其他流行的测定包括新颖槽潜水试验15和社会行为的测试，例如变浅16。下面的过程将概述的重复传送的鱼或鱼个体的群体到含有目标化合物的药理学方案的有效方法。此外，测试焦虑与光照/黑暗试验过程在鱼谁在戒断暴露于醇施用长时间表后组进行说明。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Three Shelf benchtop housing system</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/research-systems/z-hab-system/">http://aquatichabitats.com/research-systems/z-hab-system/</a></td>
		</tr>
		<tr>
			<td>1.5L Spawning tank w/400 micron baffle</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/">http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/</a></td>
		</tr>
		<tr>
			<td>Pure Grain Ethanol</td>
			<td>Luxco, INC</td>
			<td>N/A</td>
			<td><a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;aid=3759149&amp;store=Scientific">http://www.luxco.com/public/brands/brands.asp?brandid=21</a></td>
		</tr>
		<tr>
			<td>Ethovision XT Motion tracking software</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt">http://www.noldus.com/animal-behavior-research/products/ethovision-xt</a></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Canada</td>
			<td></td>
			<td><a href="http://www.eppendorf.ca/int/index.php?l=211&amp;pb=e396f9e705e754a3&amp;action=products&amp;contentid=1&amp;catalognode=9477.">http://www.eppendorf.ca/int/index.php?l=211&#38;pb=e396f9e705e754a3&#38;action=products&#38;contentid=1&#38;catalognode=9477.&#160;</a></td>
		</tr>
		<tr>
			<td>Light/Dark Arena</td>
			<td>Custom</td>
			<td></td>
			<td>Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long</td>
		</tr>
	</tbody>
</table>

a novel method of drug administration to multiple zebrafish (danio rerio) and the quantification of withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

为了保持精度和控制在药理研究与斑马鱼是很重要的时候乙醇给药的持续时间一致和准确，如上所述。我们的过程可以增加给药过程的便利性和吞吐量。对任一每周-狂欢或每日中度时间表乙醇的给药导致改变的焦虑程度，与光照/黑暗试验测定，与对照组相比。当最后一次给药后两天测试，斑马鱼，对照组（谁没有收到任何酒精，但还是被转移到配料罐）中显示的行为预期的模式，与对照鱼的消?...

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

药品监督管理局多个斑马鱼的新方法（<em&gt;斑马鱼</em&gt;）和退出的量化

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

a-novel-method-drug-administration-to-multiple-zebrafish-danio-rerio

Research

JoVE Journal

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

12.0K 次观看.  MacEwan University. A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

视频: A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

新型仪器和药物加固方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

科研

神经科学

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤：一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

慢性创伤性脑病模型的一种新方法<em&gt;果蝇</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

基于改进多平台法的大鼠中心疲劳模型

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...