这项工作一直由美国国立卫生研究院资助RO1DA014640 3R01DA014640 - 05S1，在得克萨斯研究室副总裁（CLD），奈达药品供应计划大学，得克萨斯大学的瓦戈纳中心为酒精和成瘾问题研究的布鲁斯 - 琼斯研究生奖学金（EYM） 。我们感谢他们在USV数据分析的援助莉娅McAleer，穆罕默德阿卜杜拉，Neha Thakore，拜伦巴克斯代尔，蒂芙尼阮，田甜，猎人欧文，Proy Phongsawad，海伦里德，雷切尔Chavana，琳达菊和Rosie马多克斯。

*=重要点<ol><li>精心处理的雄性Sprague - Dawley大鼠进行培训，以杠杆压与食物奖励（45毫克蔗糖颗粒;生物服务，弗伦奇敦，新泽西州）。 </li><li> *静脉导管的构造，不锈钢套管（塑料之一，弗吉尼亚州）和硅橡胶管。大鼠接受一个颈静脉导管手术过程，让药物的自我管理。手术后，导管通畅，维持每天0.1毫升0.9％生理盐水，肝素和Timentin解决方案的冲洗。 </li><li>可卡因（NIDA的药品库存，供应和控制程序; RTI国际研究三角园，数控）在本实验中使用的是溶解在适当的剂量浓度，根据动物的重量（0.75毫克/公斤/注射等渗盐水（0.9％） ），所以，每次注射0.1毫升。对照组动物在相同体积/注塑的生理盐水溶液。 </li><li> *使用药物自我管理的设备包括一个单杆操作性室配备了房子和刺激的灯光和3套photobeams（28 × 22 × 21厘米）。操作性室坐落在一个较大的声音衰减室（MED相关，圣奥尔本斯，VT）来限制外界噪音和光线。在管理会话，动物静脉通过油管连接注射器安装在机动泵含有可卡因的解决方案（0.75毫克/公斤/注射）或无菌生理盐水（Razel科学仪器，A型，圣奥尔本斯，VT） 。每个杆按下后，上面的杠杆刺激的光照亮和可卡因或生理盐水超过6秒的输液时间管理。自主活动被评定为photobeam中断。药物自我管理程序的控制，使用MED奔腾电脑配备了PC软件MED（MED联营）和行为反应（例如，杆反应和自发活动）记录。 </li><li> *检测并记录USVs，超声波麦克风，数据采集硬件，记录软件（Avisoft Bioacoustics，柏林，德国）和PC计算机系统是必要的。对于目前的实验，超声波麦克风（= 10 - 100 - kHz的频率响应范围）牢固地安装在中心的操作性商会。话筒连接到数据采集硬件（Avisoft Ultrasoundgate）位于操作性室以外的录音是在PC机上的电脑系统（戴尔Optiplex GX270，Windows XP专业版，英特尔（R）奔腾（R）4处理器，2.60 GHz的收集， 1 GB的RAM），使用录音多声道录音软件（Avisoft Bioacoustics）。 </li><li> *每种药物self-administration/USV记录如下会议所得： <ol class="lalpha"><li>动物轻轻取出他们的家庭笼，他们留置静脉留置针是用0.05毫升的一个肝素生理盐水溶液，以检测出可能的导管故障刷新。 </li><li> *老鼠，允许两种药物或车辆管理过程中的自我管理会议上的操作性室安装一个旋转连接。动物轻轻放入操作性室的大门正在悄悄地关闭。该药物的自我管理方案（MED电脑）和USV录音软件（录像机）是两个单独的计算机系统。因此，为了能够链接操作性室USV检测行为，这两项计划需要同时启动（例如，可能需要两个人，根据电脑的位置）。 </li><li> *个别USVs记录和时间戳记在整个会议期间持续。 MedPC方案，控制和记录内的操作性室的事件，可写的任何规格。 MedPC计划在会议结束时自动关闭。 USV录音需要手动停止USV录音专用计算机系统。 </li><li>在会议结束时，动物轻轻取出的操作性室，从旋转断开和刷新用0.05毫升肝素生理盐水，以清除药物残留导管。 </li></ol></li><li> *后勤用途，我们使用一个单独的计算机系统，USV分析（戴尔Optiplex 745，Windows XP专业版，英特尔（R）酷睿（TM），2个CPU，2.40千兆赫，2 GB RAM），但也可以分析软件安装在同一台计算机上的录音软件系统。 USV必须进行仔细分析，以确保准确性： <ol class="lalpha"><li> * SASLab分析软件（Avisoft Bioacoustics，柏林，德国）允许记录USVs可见和发声spectogram显示人体实验者。 </li><li> *使用可用的配置设置，导致spectogram，可以修改个人喜好。此外，可以切出低频率的背景噪音（例如，由风扇操作性商会产生），让软件自动检测高于这个频率的声音在spectogram。这些功能协助数据分析通过短，更快的声音文件，以评估。 </li><li>有时在频谱检测与USVs相同频率的背景噪音。由于声音文件。wav文件记录，研究人员需要听取在评估过程中的声音文件，以确认和区别于背景噪声USVs。 </li><li> *分析软件，可以创建Excel文件，如呼叫频率，带宽，持续时间，并发出的USV的确切时间的参数。后者允许USVs相关的确切时间之前或之后的药物是自我管理。这允许USVs用于实时测量老鼠的情绪反应在实验过程中的具体实例。 </li></ol></li></ol>代表性的成果： <img src="/files/ftp_upload/2041/2041fig1.jpg" alt="图1" /> 图1：22 kHz范围内的USV Spectogram显示 ，在22 kHz的范围内，调用与厌恶刺激引起，被认为是反映一种消极的情绪状态。 <img src="/files/ftp_upload/2041/2041fig2.jpg" alt="图2" /> 图2：Spectogram显示50千赫USVs短的“扁平化”和“调频”50千赫USVs预期和可卡因的自我管理过程中引起的。它们与积极的影响。 <img src="/files/ftp_upload/2041/2041fig3.jpg" alt="图3" /> 图3：可卡因诱导的50 kHz的范围内USVs图描绘USVs（10秒箱）之前和之后自我管理的一个代表大鼠可卡因的自我管理的第5天注射可卡因（0.75毫克/公斤/损伤）引起会议。后立即操作性室内安置和可卡因供应前，可卡因预期USVs引发和在自我管理，可卡因诱导USVs排放。

<ol>
	<li>Brudzynski, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+of+adult+rats+by+ultrasonic+vocalization:+biological,+sociobiological,+and+neuroscience+approaches.">Communication of adult rats by ultrasonic vocalization: biological, sociobiological, and neuroscience approaches.</a> ILAR J. 50 (1), 43-43 (2009).</li><li>Knutson, B., Burgdorf, J., Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+vocalizations+as+indices+of+affective+states+in+rats.">Ultrasonic vocalizations as indices of affective states in rats.</a> Psychol Bull. 128 (6), 961-961 (2002).</li><li>Wohr, M., Schwarting, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+care,+isolation-induced+infant+ultrasonic+calling,+and+their+relations+to+adult+anxiety-related+behavior+in+the+rat.">Maternal care, isolation-induced infant ultrasonic calling, and their relations to adult anxiety-related behavior in the rat.</a> Behav Neurosci. 122 (2), 310-310 (2008).</li><li>Knutson, B., Burgdorf, J., Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticipation+of+play+elicits+high-frequency+ultrasonic+vocalizations+in+young+rats.">Anticipation of play elicits high-frequency ultrasonic vocalizations in young rats.</a> J Comp Psychol. 112 (1), 65-65 (1998).</li><li>Burgdorf, J., Panksepp, 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+neurobiology+of+positive+emotions.">The neurobiology of positive emotions.</a> Neurosci Biobehav Rev. 30 (2), 173-173 (2006).</li><li>Mu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+cocaine+exposure+induces+sensitization+of+ultrasonic+vocalization+in+rats.">Repeated cocaine exposure induces sensitization of ultrasonic vocalization in rats.</a> Neurosci Lett. 453 (1), 31-31 (2009).</li><li>Ahrens, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+intravenous+amphetamine+exposure:+rapid+and+persistent+sensitization+of+50-kHz+ultrasonic+trill+calls+in+rats.">Repeated intravenous amphetamine exposure: rapid and persistent sensitization of 50-kHz ultrasonic trill calls in rats.</a> Behav Brain Res. 197, 205-205 (2009).</li><li>Burgdorf, J. <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+50-kHz+ultrasonic+vocalizations+in+rats:+electrode+mapping,+lesion,+and+pharmacology+studies.">Neurobiology of 50-kHz ultrasonic vocalizations in rats: electrode mapping, lesion, and pharmacology studies.</a> Behav Brain Res. 182 (2), 274-274 (2007).</li><li>Knutson, B., Burgdorf, J., Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonic+vocalizations+index+conditioned+pharmacological+reward+in+rats.">High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats.</a> Physiol Behav. 66 (4), 639-639 (1999).</li><li>Anagnostaras, S. G., Schallert, T., Robinson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+processes+governing+amphetamine-induced+psychomotor+sensitization.">Memory processes governing amphetamine-induced psychomotor sensitization.</a> Neuropsychopharmacology. 26 (6), 703-703 (2002).</li><li>Ferrario, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+and+behavioral+plasticity+associated+with+the+transition+from+controlled+to+escalated+cocaine+use.">Neural and behavioral plasticity associated with the transition from controlled to escalated cocaine use.</a> Biol Psychiatry. 58 (9), 751-751 (2005).</li><li>Flagel, S. B., Robinson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+psychomotor+activating+effects+of+cocaine+in+the+rat.">Quantifying the psychomotor activating effects of cocaine in the rat.</a> Behav Pharmacol. 18 (4), 297-297 (2007).</li><li>Pierce, R. C., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+behavior.">Locomotor behavior.</a> Curr Protoc Neurosci. Chapter 8 (Unit 8), 1-1 (2007).</li></ol>

大鼠发声，服务社会和communicatory的功能和表达主观的情感状态1,2的超声波频率。触发的事件，引起焦虑和苦恼，如电击线索，食肉动物的存在，厌恶药物和社会的失败2,3，22千赫USVs。因此，这些通常是长期USVs（&gt; 0.3秒）被认为是代表一种动物的负面影响状态。相反，较短的50千赫USVs（&gt; 0.3秒）发射期间和预期的正面交锋，如觅食，交配和社会发挥，就这样被链接到一个积极的情绪状态的动物 2,4 。这些调用也已加强与激活多巴胺系统5的事件相关联。例如，增加50千赫USVs已引起全身注射可卡因6或D -安非他明和以前与药物滥用8-10配对的环境。 有人曾提出，在啮齿类动物的自发活动的增加与增强的动力和食欲的行为11-14。然而，这种关系是间接的为最佳。通过研究的出现和特定频率USVs的持续，有可能获得洞察力，情绪的影响，各方面的用药经验。 在这个实验装置，我们立即收集USV录音之前和期间药物自我管理会话。这允许我们开发药物的期望，在非药物的时间间隔检查只是在会议开始之前，评估USVs可卡因的直接影响。该药物的自我管理方法使老鼠直接控制自己的药物摄入量动物和发展的时机和频率药品监督管理局准确的预期。因此，在这些会议的过程中发现的USVs反映给药准确的预期。此外，USVs可以进行评估过程中的时间间隔之前，药物的供应（例如，预期USVs），在老鼠接受实验者服用过的药物，并在药物灭绝试验。因此，在药物的预期，非然药品监督管理局和戒毒相关的影响变化也可确定。 USV变化在短期和长期的药物暴露的过程的分析，可以提供一个情感运作的药物暴露的影响更详细的解释。 除了药物自我管理实验，操作性商会USV录音，也可以用于各种奖励或厌恶的其他行为的操作，如饮酒，食品加固，并担心空调和​​灭绝的过程，。 重要的方法论详细信息由于MedPC软件可用于精确地操纵实验条件，如杆的可用性和照明条件，USV记录和分析相结合使用，允许药物相关的线索介绍，情绪的影响（即光演示文稿或其他感官刺激）摄入药物的直接影响加以确定。因此，为了确保重叠的自我管理程序和USV录音的时间表，它是至关重要的软件程序（例如，MedPC和录音机），同时被激活，在每届会议开始。 由于大量的存储USV数据（例如，一个10分钟的声音文件需要约250 MB）所需的数字空间，谨慎时，必须采取多种动物是在同一时间运行在一台计算机系统。为了避免记录错误，记录计算机系统RAM大小的需求是巨大的（1-2 GB）。其他的方式，以减少记录错误，包括在录制过程中和/或减少在同一时间运行的动物的数量降低USV决议。此外，重要的是，整个实验过程中数据收集各方面的研究人员已提高警觉。例如，大量的硬盘驱动器上的记录数据的积累可能导致USV录制节目暂停。在这种情况下，录制节目的需要，立即重新启动，以避免损失USV数据和同步与其他行为的措施USVs。 环境条件可能会影响USV调用。例如，由一个陌生的人类接触可能会导致动物发出负22 kHz的调用，这可能是误厌恶的实验条件。因此，密集的处理之前，实验操作行为实验的一个重要方面。此外，由于新颖的环境中也可引起非特异性的USV调用，动物应该是习惯于在测试环境中，如果可能的话。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Operant Conditioning Behavior (Drug Self-administration) Test Package for Rat</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>MED-008-CT-B1</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Source and Detector (Photobeams)</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-253SD ENV-253</td>
<td>&nbsp;</td>
<tr>
<td>Med PC Software</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>SOF-735</td>
<td>&nbsp;</td>
<tr>
<td>Single speed syringe pump</td>
<td>&nbsp;</td>
<td>Razel Scientific Instruments</td>
<td>Model R-E</td>
<td>&nbsp;</td>
<tr>
<td>45 mg sucrose pellets</td>
<td>&nbsp;</td>
<td>Bio-Serv</td>
<td>F0042</td>
<td>&nbsp;</td>
<tr>
<td>Catheter cannula</td>
<td>&nbsp;</td>
<td>Plastics One</td>
<td>C313G-5UP</td>
<td>&nbsp;</td>
<tr>
<td>Avisoft-UltraSoundGate 416H</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoustics</td>
<td>Part #34163</td>
<td>&nbsp;</td>
<tr>
<td>Ultrasonic microphones</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoustics</td>
<td>Part #40011</td>
<td>&nbsp;</td>
<tr>
<td>Avisoft-RECORDER Software</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoutics</td>
<td>Part #10302</td>
<td>&nbsp;</td>
<tr>
<td>Avisoft-SASLab Software</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoutics</td>
<td>Part #10101</td>
<td>&nbsp;</td>
<tr>
<td>PC Computer for USV recording</td>
<td>&nbsp;</td>
<td>Dell Optiplex GX270, Windows XP Professional, Intel (R) Pentium (R), 4 CPU, 2.60 GHz, 1 GB RAM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PC Computer for USV data analyses</td>
<td>&nbsp;</td>
<td>Dell Optiplex 745, Windows XP Professional Intel(R) Core (TM), 2 CPU, 2.40 GHz, 2GB RAM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cocaine</td>
<td>&nbsp;</td>
<td>RTI International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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assessment of ultrasonic vocalizations during drug self-administration in rats

常用药物自我管理程序研究与人类滥用药物成瘾和复吸相关的行为和神经化学变化。通常用作药物的激励措施，在动物行为活动的各类。然而，药物滥用戒断可卡因使用者复发的一个重要组成部分，是“渴求”，这是很难在动物模型，因为它经常在公开行为的情况下发生。然而，它可能是一类超声波发声大鼠（USVs）药品监督管理局，药品的期待和渴求的情感反应可能是有用的标记。大鼠作为一个communicatory功能和表达主观的情感状态的超声波频率发声。一些研究表明，不同的呼叫的频率范围是消极和积极的情绪状态有关。例如，高频电话（“50千赫”）与积极的影响，而低频率的呼叫（“22千赫）代表一种消极的情绪状态。本文介绍的程序，以评估与日常可卡因自我管理相关的大鼠USVs。在此过程中，我们利用标准的单杆安置内操作性商会声音衰减箱可卡因的自我管理的会议，并利用超声波麦克风，多通道录音硬件和专门的软件程序来检测和分析USVs。 USVs测量大鼠情感反映之前，期间和之后，药物的供应，可与常用评估药物自我管理行为数据等杠杆的反应，反应间的时间间隔和自主活动相关。由于USVs可以在间隔之前，药物的供应（例如，预期USVs），并在药物灭绝试验中影响评估，与预期毒品和戒毒相关的变化也可以。此外，确定USV的变化在短期和长期的药物暴露的过程，可以提供一个情感运作的药物暴露的影响更详细的解释。

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Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

药物自我管理和超声波发声（USV）用作动物研究中的行为评估，但很少结合。本文的目的是在药物的自我管理程序描述记录USVs的用药经验的优势，以评估情感反应。

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 ...

assessment-ultrasonic-vocalizations-during-drug-self-administration

Research

JoVE Journal

Neuroscience

17.3K 次观看.  University of Texas at Austin. 药物自我管理和超声波发声（USV）用作动物研究中的行为评估，但很少结合。本文的目的是在药物的自我管理程序描述记录USVs的用药经验的优势，以评估情感反应。

视频: Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

老年帕金森病大鼠的超声波发声的有针对性的培训

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

科研

神经科学

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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.  ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

皮质脊髓束的大鼠单侧锥体束的神经可塑性诱导疗法的评估

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

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 ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

单边坐骨神经粉碎损伤后大鼠运动行为的多因素评估

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

同时记录脑电图的大鼠脑脊液和血液

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

作者聚焦：获取高质量的 CSF 和血液样本以发现癫痫生物标志物 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...