这个手稿和视频的生产，支持由国家药物滥用/国家卫生研究院授予R15的DA016285 - 02，西华盛顿大学生物医学在神经科学倡议的研究活动，研究所和西华盛顿大学。

1。受试动物<ol><li>男子跳远- Evans大鼠在研究开始3个月大。老鼠西蒙森衍生（美国，加利福尼亚州，吉尔罗伊）在西华盛顿大学vivarium的培育和个别被安置在12 - H反日/夜循环普瑞纳米尔斯公司Mazuri啮齿动物颗粒（灰色首脑会议（0700灯光熄灭）美国密苏里州）和水可用自由采食。 </li><li>所有的培训和测试之间需要0900-1300的地方总是每天在同一时间的训练和测试大鼠的同伙。 </li><li>老鼠权衡研究期间每星期一，星期三和星期五。 </li><li>紧接训练阶段，老鼠被剥夺约20小时的水，以鼓励蔗糖的自我管理训练的第一天。 </li></ol> 2。行为程序所有操作性行为程序使用标准的操作条件钱伯斯（MED Associates公司，圣奥尔本斯VT，美国）的每个操作条件室顶部输液泵（Razel科学仪器，圣奥尔本斯，佛蒙特州，美国）。操作性条件反射室是在封闭的声音衰减换气扇柜（西华盛顿大学，美国）。第四MED电脑软件（MED Associates公司，圣奥尔本斯，佛蒙特州，美国）控制所有的实验条件和数据收集。 操作性条件反射室（30 × 20 ​​× 24厘米），包含两个杠杆（一个固定和一个可伸缩），音频发生器，白刺激光以上的伸缩杆，和对面的墙壁上的红房子光。输液泵可为蔗糖的奖励插座的积极杠杆的权利。杠杆从格地板10厘米。四个红外photobeams纵横交错，每个分庭。前束是从每个侧墙10.5厘米，侧梁侧墙每6厘米。每束4.5厘米以上的不锈钢棒地板。梁一个重新设置为计数完整的休息。梁断裂的总数都记录在培训和渴望测试。 除限水前的第一次训练，老鼠提供的食物和水，无论是在家里笼子里，在操作性条件反射腔随意。钱伯斯是在一个专门的试验室，与vivarium反向光周期。老鼠运家笼之间的推车上的挡光布覆盖的客房。在每届会议结束时，老鼠返回首页笼。 典型的实验：训练，被迫禁欲，和测试分为三个阶段。测试是评估会议其中渴求。我们评估在两个一般的测试程序的渴求，取决于研究的。这两个过程的描述在第2.3.1和2.3.2。 <ol><li>培训<ol><li>在典型的实验中，大鼠花2小时/天操作条件，允许他们按杆右侧的插座交付了10％的蔗糖溶液0.2毫升到的伸缩杆（活动）商会连续10天。 </li><li>这种反应也激活了一个音（2 kHz时，环境噪音超过15分贝）和白光组成的复合刺激。复合刺激持续时间为5秒，其次是一个40的时间，在此期间，印刷机上的积极杠杆被记录，但有没有编程的后果。 </li><li>一个无效（固定）杠杆的反应，没有编程的后果，但印刷机记录。梁断裂的数字记录在训练和测试。随着时间的课程（2分钟箱）记录所有的反应和运动措施，但通常是作为每节的总计报道。 </li></ol></li><li>强制禁欲。大鼠被迫禁欲时间留在家里笼子。我们使用典型的被迫禁欲期间有老鼠测试日发送一天后立即训练的最后一天（被迫禁欲的第1天）或30天以下的培训（被迫禁欲30日）的最后一天。 </li><li>测试<ol><li>消无线索。这个测试过程中，老鼠是第一个获准按异常反应没有后果，并在会议期间在条件相同的培训的积极杠杆是1小时大鼠经验，在考试当天至少6这些灭绝会议。每个分隔活动杆收回房子的灯是关闭其中一个5分钟的时间内灭绝会议。 “灭绝”表示，如果有源杆响应已经减少到不到20压机/小时。有些老鼠需要第七届会议。下届会议（另外5分钟的延迟后）是测试会议​​，大鼠可以为基调，然后按+光提示，最初是在培训期间，每个蔗糖交付配对。 </li><li>消与线索。在此缩写ð测试过程中，老鼠被允许按条件相同，但蔗糖不交付训练活动的杠杆。 </li></ol></li><li>无效杆紧迫的是用来作为衡量杠杆歧视。同时，这也是photobeam休息，作为衡量一个非特定的电机激活。肌肉运动与动机的变化复杂的渴求行为的解释。不活动的杠杆响应photobeam打破数据是有用的澄清电机与动力贡献的渴求，是在回应率很高，在蔗糖或其他食品的老鼠是一个潜在的渴望影响复合的挑战的药理研究的对照组（如7 8）。 </li></ol> 3。代表操作，影响到后强制禁欲可变时期的蔗糖的渴望这里描述的一般方法提供了一种方法来探测Determina的行为和神经生物学NTS后被迫禁欲的变量时期渴望。这里描述的方法代表性的结果在第4节提供有关。 <ol><li>饱食。一些大鼠瓶在自己的笼子里的蔗糖10％，17小时前测试8。在这项研究中大鼠进行了测试，没有线索的过程中使用的灭绝。 </li><li>丰富环境。在个人住房（对照组），而其他人则成一个大移动（36“宽x 20”深x 40“的高度被迫禁欲期期间，还存在着一些大鼠笼产品质量，波特兰，OR，美国）与三个同伙笼（丰富）。富集的动物曾在其环境中的几个玩具，包括PVC管材，也给予新奇玩具每一个M，W，F 10，在这项研究中的大鼠进行了测试，没有线索的过程中使用的灭绝。 </li><li>药理挑战。测试当天，大鼠预处理剂量的多巴胺D1拮抗剂SCH 23390。大鼠在这项研究中WER发送测试与线索的过程中使用的灭绝。 </li></ol> 4。代表性的成果： <img alt="图1" src="/files/ftp_upload/3335/3335fig1.jpg" /> 图1代表的培训和孵化蔗糖渴求实验的测试数据。 数据点显示手段±中小企业。图1a组的大小是77大鼠和图1B组大小从11-12％不等组。 *显著的差异，从第1天组，P &lt;0.05。这个数字是改编自8施普林格的许可数据。 <img alt="图2" src="/files/ftp_upload/3335/3335fig2.jpg" /> 图2蔗糖渴求的影响蔗糖饱食后1或被迫禁欲30天。组大小为每组8-9。 *显著的差异，从第1天组，†显着性差异对照组，P &lt;0.05。此图是适应从9与爱思唯尔的权限。 <img alt="图3" src="/files/ftp_upload/3335/3335fig3.jpg" /> 图3丰富环境一个月减少蔗糖的渴求。组大小各8只。 *从第一天开始，†从对照组，P &lt;0.05显着性差异显着性差异。这个数字是适应从10威科卫生许可。 <img alt="图4" src="/files/ftp_upload/3335/3335fig4.jpg" /> 图4全身中学更有效的后一天被迫禁欲23390减少蔗糖的渴求。组大小为8-12每个组。 *从第1天组，†0剂量组，P &lt;0.05的显著差异显着性差异。这个数字是改编自8施普林格许可。 <ol><li>图1表明大鼠，自我管理的蔗糖代表的训练和测试数据（图1顶部），然后与蔗糖配对提示（图1底部）灭绝的响应。如上所示，大鼠回应选择性地在训练和在测试过程中的积极杠杆。有趣的是，不活动的响应，并自发活动表现出一定的潜伏期，虽然效果是最强大的积极响应杆的。 </li><li>图2显示了饱食后对蔗糖的渴求蔗糖1或被迫禁欲30天的效果。饱食减少无响应线索后，无论是被迫禁欲期间灭绝，但只减少响应蔗糖线索仅只有一个被迫禁欲天的大鼠。 </li><li>正如图3所示，环境富集减少孵育灭绝，没有提示的响应，并单独为蔗糖的提示响应行为只有一个被迫禁欲一天后发现的水平。 </li><li>图4描绘了一个线索响应灭绝中学23390禁欲依赖效应。中学23390最EFfective在减少后1蔗糖的渴求，与30天被迫禁欲。 </li></ol>

<ol>
	<li>Sobik, L., Hutchison, K., Craighead, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cue-elicited+craving+for+food:+a+fresh+approach+to+the+study+of+binge+eating.">Cue-elicited craving for food: a fresh approach to the study of binge eating.</a> Appetite. 44, 253-2561 (2005).</li><li>Geiger, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+of+mesolimbic+dopamine+neurotransmission+in+rat+dietary+obesity.">Deficits of mesolimbic dopamine neurotransmission in rat dietary obesity.</a> Neuroscience. 159, 1193-1199 (2009).</li><li>Grimm, J. W., Olmstead, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Craving In Animal Models of Drug Addiction. , 311-336 (2011).</li><li>Grimm, J. W., Kruzich, P. J., See, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contingent+access+to+stimuli+associated+with+cocaine+self-administration+is+required+for+reinstatement+of+drug-seeking+behavior.">Contingent access to stimuli associated with cocaine self-administration is required for reinstatement of drug-seeking behavior.</a> Psychobiology. 28, 383-386 (2000).</li><li>Bossert, J. M., Ghitza, U. E., Lu, L., Epstein, D. H., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+relapse+to+heroin+and+cocaine+seeking:+an+update+and+clinical+implications.">Neurobiology of relapse to heroin and cocaine seeking: an update and clinical implications.</a> Eur. J. Pharmacol. 526, 36-50 (2005).</li><li>Bossert, J. M., Poles, G. C., Wihbey, K. A., Koya, E., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+blockade+of+dopamine+D1-family+receptors+in+nucleus+accumbens+core+or+shell+on+reinstatement+of+heroin+seeking+induced+by+contextual+and+discrete+cues.">Differential effects of blockade of dopamine D1-family receptors in nucleus accumbens core or shell on reinstatement of heroin seeking induced by contextual and discrete cues.</a> J. Neurosci. 27, 12655-12663 (2007).</li><li>Grimm, J. W. <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+systemic+or+nucleus+accumbens-directed+dopamine+D1+receptor+antagonism+on+sucrose+seeking+in+rats.">Effects of systemic or nucleus accumbens-directed dopamine D1 receptor antagonism on sucrose seeking in rats.</a> Psychopharmacology (Berl). , (2011).</li><li>Grimm, J. W., Fyall, A. M., Osincup, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+sucrose+craving:+effects+of+reduced+training+and+sucrose+pre-loading.">Incubation of sucrose craving: effects of reduced training and sucrose pre-loading.</a> Physiol Behav. 84, 73-79 (2005).</li><li>Grimm, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+attenuates+cue-induced+reinstatement+of+sucrose+seeking+in+rats.">Environmental enrichment attenuates cue-induced reinstatement of sucrose seeking in rats.</a> Behav Pharmacol. 19, 777-785 (2008).</li><li>Shaham, Y., Shalev, U., Lu, L., De Wit, H., Stewart, 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+reinstatement+model+of+drug+relapse:+history,+methodology+and+major+findings.">The reinstatement model of drug relapse: history, methodology and major findings.</a> Psychopharmacology (Berl). 168, 3-20 (2003).</li><li>Childress, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbic+activation+during+cue-induced+cocaine+craving.">Limbic activation during cue-induced cocaine craving.</a> Am J Psychiatry. 156, 11-18 (1999).</li><li>Gearhardt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Correlates+of+Food+Addiction.">Neural Correlates of Food Addiction.</a> Arch. Gen. Psychiatry. , (2011).</li><li>Markou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+drug+craving.">Animal models of drug craving.</a> Psychopharmacology (Berl). 112, 163-182 (1993).</li><li>Phillips, R. G., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+amygdala+and+hippocampus+to+cued+and+contextual+fear+conditioning.">Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning.</a> Behav. Neurosci. 106, 274-285 (1992).</li><li>Holland, P. C., Bouton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampus+and+context+in+classical+conditioning.">Hippocampus and context in classical conditioning.</a> Curr. Opin. Neurobiol. 9, 195-202 (1999).</li><li>Epstein, D. H., Preston, K. L., Stewart, J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+model+of+drug+relapse:+an+assessment+of+the+validity+of+the+reinstatement+procedure.">Toward a model of drug relapse: an assessment of the validity of the reinstatement procedure.</a> Psychopharmacology (Berl). 189, 1-16 (2006).</li><li>Kupferschmidt, D. A., Brown, Z. J., Erb, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Procedure+for+Studying+the+Footshock-Induced+Reinstatement+of+Cocaine+Seeking+in+Laboratory+Rats.">A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats.</a> J. Vis. Exp. (47), (2011).</li></ol>

所有程序中的“实验动物保健的原则”（NIH出版物。86-23）所述的指引和西华盛顿大学机构动物护理和使用委员会批准。

我们在这里描述的程序可确定为恢复程序，虽然这在技术上是不准确的杆按提示是没有熄灭前的渴求测试会议。在一个典型的恢复过程，响应与奖励配对线索熄灭，然后选择“恢复”与管理的奖励（吸诱导复职）非然介绍，或如电击应激（应激诱导复职）11 。杆按下单独访问的提示（项目2.3.1）的灭绝是最接近复职。然而，我们所描述的程序，允许测量目标导向的蔗糖配对提示的响应。操...

<table border="1"><tbody><tr><td>项目</td><td>公司</td><td>目录编号</td></tr><tr><td>富集笼</td><td>质量凯奇公司</td><td> FH - 36半的T - ñ </td></tr><tr><td>操作性商会</td><td> MED联营公司</td><td> MED - 008 - CT - B3 </td></tr><tr><td>中学23390 </td><td>西格玛</td><td> D054 </td></tr></tbody></table>

a general method for evaluating incubation of sucrose craving in rats

有关的食物限制饮食，在应对食品配对的线索食物渴求的人，可作为行为禁欲和复发的食物，服用1之间一个关键的转型点。食物的渴求在这样的概念是类似毒品的渴求反应的药物配对线索。丰富的文献一直围绕了解药物渴求的行为和神经生物学因素，我们和其他人最近一直专注于翻译的基本成瘾研究的技术，以便更好地了解有关食品2-4毒瘾般的行为。 在以往的研究药物渴求中所做的那样，我们利用大鼠模型的复发研究蔗糖的渴求行为。在这种模式下，老鼠的自我管理超过数天两种药物在会话或食物。在一个会话中，杆响应，提供奖励，伴随着一个基调+光刺激。渴望的行为，然后运作定义为在一个响应随后举行的会议，奖励是不可用。大鼠将可靠地响应为基调+光刺激，可能是由于其收购的空调补强性能 5 。这种行为有时也被称为蔗糖寻求或暗示反应。在目前的讨论中，我们将使用术语“蔗糖渴望”归入这两个构造。 在过去的十年中，我们主要集中在以下奖励自我管理的影响奖励渴求的时间长度如何。有趣的是，老鼠在几个星期的强制禁欲期过程中增加奖励配对提示响应。这种“渴望孵化”观察大鼠，自我管理无论是食品或药物滥用 4,6 。这在渴望的时间依赖性的增加，我们已经在动物模型确定人类药物和食品成瘾行为可能有很大的潜在的相关性。在这里，我们提出了一个协议，评估sucro孵化SE大鼠的渴求。变种程序将显示渴求评估是作为一个独立的蔗糖配对提示，内蔗糖的自我管理的范围内（无线索灭绝）或响应蔗糖配对暗示在一般的灭绝背景下按以下的杠杆灭绝响应（线索灭绝）。

Watch this Scientific Journal Video about A General Method for Evaluating Incubation of Sucrose Craving in Rats at JoVE.com

A General Method for Evaluating Incubation of Sucrose Craving in Rats

响应食物或药物配对暗示过一段时间的禁欲过程中增加，这可能与复发行为的易感性增加。在这里，我们详细评估在大鼠体内有自我管理的蔗糖的这个“孵化渴求”的程序。

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

a-general-method-for-evaluating-incubation-of-sucrose-craving-in-rats

Research

JoVE Journal

Neuroscience

13.2K 次观看.  Western Washington University. 响应食物或药物配对暗示过一段时间的禁欲过程中增加，这可能与复发行为的易感性增加。在这里，我们详细评估在大鼠体内有自我管理的蔗糖的这个“孵化渴求”的程序。

视频: A General Method for Evaluating Incubation of Sucrose Craving in Rats

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

一个简单的综合评分系统评估小脑共济失调的小鼠模型的表型

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

科研

神经科学

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

粉条和Capellini处理测试：在大鼠和小鼠的灵巧的前爪功能简单的定量措施

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Touchscreen Sustained Attention Task (SAT) for Rats

Sustained attention is the ability to monitor intermittent and unpredictable events over a prolonged period of time. This attentional process subserves other aspects of cognition and is disrupted in certain neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Thus, it is clinically important to identify mechanisms that impair and improve sustained attention. Such mechanisms are often first discovered using rodent models. Therefore, several behavior procedures for testing aspects of sustained attention have been developed for rodents. One, first described by McGaughy and Sarter (1995), called the sustained attention task (SAT), trains rats to distinguish between signal (i.e., brief light presentation) and non-signal trials. The signals are short and thus require careful attention to be perceived. Attentional demands can be increased further by introducing a distractor (e.g., flashing houselight). We have modified this task for touchscreen operant chambers, which are configured with a touchscreen on one wall that can present stimuli and record responses. Here we detail our protocol for SAT in touchscreen chambers. Additionally, we present standard measures of performance in male and female Sprague-Dawley rats. Comparable performance on this task in both sexes highlights its use for attention studies, especially as more researchers are including female rodents in their experimental design. Moreover, the easy implementation of SAT for the increasingly popular touchscreen chambers increases its utility.

Touchscreen Sustained Attention Task SAT for Rats

Sustained attention, or continuously monitoring situations for intermittent and unpredictable events, is a critical aspect of cognition. Here we detail how to test sustained attention in rats using touchscreen operant chambers. We demonstrate comparable performance in male and female rats, making this task useful for studying attention in both sexes.

触摸屏持续注意任务 (SAT) 大鼠

Sustained attention is the ability to monitor intermittent and unpredictable events over a prolonged period of time. This attentional process subserves ...