这项研究已经得到了爱德蒙和莉莉·萨夫拉中心脑科学（SB奖学金），以色列科学基金会（批准号：378/11，HS）的遗产遗产生物医学科学合作计划和德国以色列科学基金会研究与发展（GIF）（批准号：1093-32.2/2010 HS）。

1。慢病毒载体的制备<ol><li> HEK-293FT细胞增长到90％汇合。 </li><li>上的天的的变动转染细胞培养基中无血清DMEM辅以用1mM谷氨酰胺和50 mg / ml的青霉素 - 链霉素。 </li><li>共同转染的的细胞与pLKO.1迪普罗载体有质粒的编码为ΔR8.2和VSV-G部分和感兴趣的miRNA，用10微升的1毫克/毫升的聚乙烯亚胺作为载体9。 </li><li>在24小时和48小时后转染，收集打包慢病毒过滤0.45微米的过滤器。 </li><li>利用超速离心70,000 XG浓缩慢病毒，2小时和15℃，分装和储存在-70°C </li><li>测量病毒滴度感染的HEK-293T细胞与系列稀释的病毒制剂（载体，每孔1至10 -6毫升）。由此产生的滴度（最少〜1×10 9个感染性颗粒每毫升建议）评价表达锗NE的利益使用嘌呤霉素筛选，并通过量化表达GFP的病毒通过荧光细胞计数。 </li></ol>对于更详细的协议慢病毒制剂看到10。 2。准备进行手术的动物<ol><li>外科医生的装束应包括手术衣，无菌手套，帽子和口罩。 </li><li>称重动物，然后麻醉与IP-注射氯胺酮的混合物，与体积成比例的指定每种药物的动物体重。对于氯胺酮/赛拉嗪的混合物，剂量范围为80〜200毫克/千克氯胺酮和7-20毫克/公斤甲苯噻嗪通常用于老鼠。 </li><li>等待，直到动物完全麻醉，然后检查缺乏撤退反射的动物后肢延伸肢体，然后用手指按它。 </li><li>注入动物皮下Rimadyl，包装盒上的指定，止痛。典型剂量范围Rymadil的是5-10毫克。 </li><li>将动物加热垫，以保持稳定的体温，用膏滋润眼睛。 </li><li>剃须两个耳朵之间的区域，用修剪机。 </li><li>用优碘消毒皮肤。 </li></ol> 3。暴露的头骨和钻井<ol><li>放置的动物内stereotact通过调整杆只是前动物的耳朵缝里。 </li><li>使用一把手术刀使前后切口1.5厘米左右两耳之间，并保持它打开手术钳（serfines）的。 </li><li>清洁棉签直到冠状缝和矢状缝之间的交集， 即前囟门和日冕之间的交集和人字缝， 也就是 lambda，头骨表面是可见的。 </li><li>指向的注射器的前端，由stereotact持有，前囟点，在所有三个轴。写下的坐标。这点会被视为零婆int的所有三个轴。 </li><li>提起注射器在垂直轴上，使平面运动不会划伤的头骨和注射器头移动到正确的位置。海马CA1区注射要求以下坐标相对毫米前囟门：-2，前/后的轴，±1.8和-1.5背/腹轴的横向/中轴。 </li><li>放下注射器的尖端，直到它触及的头骨和标记的一个标记点​​。取下注射器，以避免刺伤。 </li><li>钻一个浅洞，只能用细钻颅骨。保持稳定，所以它不会继续深入到软组织下面钻。可以防止这种稳住一方面与其他钻在很短的脉冲。 </li></ol> 4。慢病毒注射液<ol><li>取出0.5毫升浓缩的慢病毒溶液。 </li><li>将注射器上面的孔，慢慢降低它垂直，直到它REACHES头骨表面。继续以降低注射器进入大脑非常缓慢。在此步骤中的一些可能发生的出血并不一定表示一个失败的渗透。如果发生出血拖把用棉签。 </li><li>数字泵以0.02毫升/分钟（0.5微升，在25分钟内，将注入）设置，并开始输液。缓慢输注允许进行有效的病毒蔓延到组织中，并防止回流。在某些类型的注射器，注射器插入一个额外的深度为0.5毫米，前撤退到原来的坐标和泡制。 </li><li>输液结束后等待5分钟，使材料扩散到大脑，而不是撤退到由注射器管形成。 </li><li>取下注射器非常缓慢，并观看回流。如果在此步骤中观察到回流，注射材料的一小部分可能丢失。 </li></ol> 5。伤口密封和恢复<ol><li>密封THÉ伤口组织黏合剂。务必到避免泄漏histoacryl，进入眼睛。 </li><li>注入动物腹腔（IP），预热生理盐水1毫升，以避免脱水。 </li><li>将恢复笼中的动物，其定位在加热垫。关注的动物，而另外1小时恢复。 </li></ol>消瘦，食欲不振的情况下，无力/无法获得饲料或饮水中，濒死状态或感染的动物实施安乐死应该被执行。啮齿动物安乐死的可接受的方法是巴比妥类药物，吸入麻醉剂，CO 2，CO，或氯化钾与全身麻醉。 4〜6周后，评估动物的导航内存在Morris水迷宫中的评估。慢病毒通常会感染大多数细胞内注射球体。 6。在Morris水迷宫行为评估<ol><li>训练动物在M鸢尾水迷宫。对于确切的训练和测试协议，见11。在每次试验之后，用干毛巾擦干动物，取代它在笼子里，并刷新水箱中的水。 </li><li>连续3天，4项试验在Morris水迷宫测试动物的每一天。每天进入迷宫的4个方向的迷宫（北，南，东，西），在不断变化的顺序在每个插入的动物，例如一天：东西南北，日二：西部南北北东。每次试验后擦拭动物。 </li><li>在试验过程中试验跟踪Noldus跟踪系统的跟踪系统，例如动物。 </li><li>经过12 次的测试试验，插入动物到水箱一分钟而不平台。这将用于分析动物服用的搜索策略。 </li><li>分析数据12中所建议的。 </li></ol>

<ol>
	<li>Siegel, 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=A+functional+screen+implicates+microRNA-138-dependent+regulation+of+the+depalmitoylation+enzyme+APT1+in+dendritic+spine+morphogenesis.">A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis.</a> Nature Cell Biology. 11, 705-716 (2009).</li><li>Jin, P., 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=Biochemical+and+genetic+interaction+between+the+fragile+X+mental+retardation+protein+and+the+microRNA+pathway.">Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway.</a> Nature Neuroscience. 7, 113-117 (2004).</li><li>Simon, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-1+regulates+a+MEF-2-dependent+retrograde+signal+at+neuromuscular+junctions.">The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions.</a> Cell. 133, 903-915 (2008).</li><li>Consiglio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+therapy+of+metachromatic+leukodystrophy+by+lentiviral+vectors:+correction+of+neuropathology+and+protection+against+learning+impairments+in+affected+mice.">In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.</a> Nature Medicine. 7, 310-316 (2001).</li><li>Jakobsson, J., Lundberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+for+use+in+the+central+nervous+system.">Lentiviral vectors for use in the central nervous system.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 13, 484-493 (2006).</li><li>Berson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic-associated+loss+of+hnRNP-A/B+in+Alzheimer's+disease+impairs+cortical+splicing+and+cognitive+function+in+mice.">Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice.</a> EMBO Molecular Medicine. , (2012).</li><li>Haramati, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+as+repressors+of+stress-induced+anxiety:+the+case+of+amygdalar+miR-34.">MicroRNA as repressors of stress-induced anxiety: the case of amygdalar miR-34.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 31, 14191-14203 (2011).</li><li>Shaltiel, 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=Hippocampal+microRNA-132+mediates+stress-inducible+cognitive+deficits+through+its+acetylcholinesterase+target.">Hippocampal microRNA-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target.</a> Brain Structure &amp; Function. , 10-1007 (2012).</li><li>Boussif, O., 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=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 7297-7301 (1995).</li><li>Mendenhall, A., Lesnik, J., Mukherjee, C., Antes, T., Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+HIV-+or+FIV-based+lentivector+expression+constructs+&amp;+transduction+of+VSV-G+pseudotyped+viral+particles.">Packaging HIV- or FIV-based lentivector expression constructs &amp; transduction of VSV-G pseudotyped viral particles.</a> J. 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作者宣称，他们有没有竞争经济利益。

立体定向注射慢病毒是一个相对快速的方法，无论是向上或向下调节不同的基因和miRNA 在体内评估。主要的替代是一个遗传工程小鼠，这是一个更费时费力的技术，然后慢直喷。此外，向上调节，在慢病毒注射，在成年小鼠发生在一个特定的时间，并且不包括任何可能的泄漏在开发过程中，是最经常的情况下，包括时间控制遗传工程小鼠。不同的基因在神经退行性疾病中的调控改变的动物?...

miRNA在神经系统功能的作用，尤其是最近已经慢病毒注射在几项研究中的挑战。已发现miRNA的维护和再塑造突触结构参数1 2，突触发生和突触重塑和维护3是至关重要的。这些研究有力地表明，miRNA的啮合时，通过多层次的调节作用，在形成和维持神经系统的主要输出，认知功能。立体定向注射慢病毒颗粒可以搜索到特定地区的啮齿类动物的大脑突触形态的改变和神经元的活动，并用于建立过表达的转录4,5的功能意义。义脑区的神经元与miRNA的表达慢病毒直接感染可能受到牵连老化，脑部疾病和神经退行性疾病的研究; miRNA的研究在行为调节6-8的境界是在一个远远低于先进国家，立体注射的miRNA表达慢病毒颗粒，然后通过行为测试等用途可能是有用的。诱导过度或不足表达相当费力的方法涉及遗传工程连锁或基因敲除小鼠。遗传系统可以进一步允许表达的条件和时间的控制（ 例如 ，春节系统CRE-LOX），但很难提供空间特异性注射过程几乎总是有一定量的泄漏。此外，工程程序并不需要手术治疗，可用于在实验室比较好的重现性，但是，他们是慢，需要更多的人力和财力。此外，时间控制的过表达小鼠注射准确得多遗传工程小鼠相比。

<table border="0" cellpadding="0" cellspacing="0" width="748">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21">Rodent weigh scale</td>
			<td>Burtons (UK)</td>
			<td>115-455</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="24">
			<td height="24">heating pad</td>
			<td>FIRstTechnology</td>
			<td>DCT-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">trimming machine</td>
			<td>Stoelting</td>
			<td>51465</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">stereotact</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Scalpel and blades</td>
			<td>Kent scientific</td>
			<td>INS500348</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Harland syringe</td>
			<td>Hamilton</td>
			<td>7632-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">driller</td>
			<td>Stoelting</td>
			<td>51449</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">digital pump</td>
			<td>Harvard apparatus</td>
			<td>704507</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Water tank and platform</td>
			<td>Stoelting</td>
			<td>60135</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0" width="682">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr height="21">
			<td height="21">ketamine </td>
			<td>Vetoquinol(Lure France)</td>
			<td>3055503</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">domitor</td>
			<td>Orion pharma</td>
			<td>107140-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Rimadyl</td>
			<td>Pfizer animal health</td>
			<td>24751</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="118">
			<td height="138" rowspan="2">moisture ointment - Synthomycine 5%</td>
			<td rowspan="2">Rekah Pharmaceutical</td>
			<td rowspan="2">195</td>
			<td rowspan="2"> </td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="21">
			<td height="21">histoacryl</td>
			<td>Braun</td>
			<td>112101</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">saline</td>
			<td>Sigma Aldrich</td>
			<td>D8662</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome

小分子RNA（miRNA）是监管小单约22个核苷酸长的双链RNA分子可能每个目标众多的mRNA转录和暗淡的整个基因表达通路诱导杀伤性和/或抑制翻译这些目标。一些miRNA的发挥关键作用，在维持神经元的结构和功能，并在更高级别的脑功能，寻求探索这些功能操作水平和方法。在这里，我们提出了一种直接在体内的方法强迫立体定向注射miRNA的编码病毒颗粒在小鼠体内多余的miRNA研究的认知后果。具体来说，目前的协议还包括海马CA1区，这有助于哺乳动物记忆的巩固，学习和应激反应，并提供了一​​个方便的注射部位注入。坐标测量，根据鼠标的前囟门和数字控制病毒的灌注，并保持很慢。注射后，手术伤口是密封的，动物的恢复。相应的靶mRNA编码消音服务慢病毒牵连特定miRNA /靶相互作用负责观察效果，​​天真的小鼠，小鼠注射用生理盐水与“空”的慢病毒载体作为对照组小鼠注射。一个月后注射，动物们检查Morris水迷宫（MWM）评估其导航的学习和记忆能力。 MWM是圆罐，充满了彩色水淹没水面1厘米以下的小平台。周围的坦克的稳态视觉线索允许空间导航（声音和地球磁场也可协助动物在导航）。视频摄像监控，使测量游的路线和时间，找到并达到平台。先教鼠标，安装隐藏的平台，提供了一个逃离强迫游泳，然后测试使用这种转义和f值为，该平台将被删除，探针测试检查，如果鼠标记得它以前的位置。连续几天的反复测试，突出改善性能测试小鼠在更短的延迟，是寻找和挂载平台，更直接的路线到达平台或位置。没有这样的代表减值改善学习记忆和/或焦虑，然后可以专门测试（ 如在高架十字迷宫）。这种方法使验证特定的miRNA在所研究的认知和/或压力相关的进程和目标成绩单。

0.5微升慢病毒注射到小鼠海马CA1区的协议部分中的指示的流量，产生约1毫米的喙尾椎轴受感染的球体，大约0.5mm的内侧 - 外侧和前后的轴（ 图3）。 <img alt="图1" src="/files/ftp_upload/50170/50170fig1.jpg" /> 图1。前囟门点和喷射装置 。 （ 一 ）所示是一个示意图，地图的前囟门（冠状面和矢状面的缝合线交汇）和L...

Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome at JoVE.com

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

小分子RNA在大脑的结构和功能有显着的作用。在这里，我们描述了一种方法来执行海马miRNA的过表达一个精心设计的miRNA表达慢病毒采用立体定向注射。这种方法可以作为一个相对快速的方式评估<em&gt;在体内</em&gt;过表达miRNA在特定的大脑区域的影响。

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

stereotactic-injection-microrna-expressing-lentiviruses-to-mouse

Research

JoVE Journal

Behavior

22.6K 次观看.  The Hebrew University of Jerusalem. 小分子RNA在大脑的结构和功能有显着的作用。在这里，我们描述了一种方法来执行海马miRNA的过表达一个精心设计的miRNA表达慢病毒采用立体定向注射。这种方法可以作为一个相对快速的方式评估在体内过表达miRNA在特定的大脑区域的影响。

视频: Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

小鼠疾病模型的集成行为车站行为表型（INBEST）

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

科研

行为学

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

衰老小鼠前庭系统的行为评估

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

转录动力学的脑组织样本在行为经验综合分析

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

使用自动图像处理方法鼠标社交性的实验评估

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

迷走神经刺激作为一种工具来诱导可塑性相关途径灭绝学习

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human auditory development for these age groups. This paper describes an observer-based procedure for measuring hearing sensitivity with a two-interval, two-alternative forced-choice paradigm. Young children are trained to perform a play-based, motor response (e.g., putting a block in a bucket) whenever they hear a target signal. An experimenter observes the child&#39;s behavior and makes a judgment about whether the signal was presented during the first or second observation interval; the experimenter is blinded to the true signal interval, so this judgment is based solely on the child&#39;s behavior. These procedures were used to test 2 to 4 year-olds (n = 33) with no known hearing problems. The signal was a 1,000 Hz warble tone presented in quiet, and the signal level was adjusted to estimate a threshold corresponding to 71%-correct detection. A valid threshold was obtained for 82% of children. These results indicate that the two-interval procedure is both feasible and reliable for use with toddlers and preschoolers. The two-interval, observer-based procedure described in this paper is a powerful tool for evaluating hearing in young children because it guards against response bias on the part of the experimenter.

This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child&#39;s behaviors.

2听证会，以4岁儿童的行为评估：两区间，观测为基础的应用程序基于条件性播放响应

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

一种用于小鼠运动技能、情绪和认知重复评估的行为测试电池

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

利用可重构的迷宫系统提高啮齿动物空间导航测试的可重复性

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

使用行为测试验证 Angelman 综合征的小鼠模型

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

作者聚焦：用于验证 Angelman 综合征小鼠模型的一系列高度可重复的行为测试 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

安格曼综合征小鼠模型的行为表征

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...