由美国国立卫生研究院（NIDCD，P30 - DC010754，T32 - DC009401）支持。

1。前/后培训记录<ol><li>使用图2所示的配置与Avisoft录像机开始超声监测。 </li><li>超声波发声可以监测直观地观看实时的频谱图，或用耳机听觉插入超声录音接口。 </li><li>将性接受的（在发情期）进入雌性大鼠受雄性大鼠的家庭笼。发情的迹象包括前凸，摆动耳朵，和快速穿梭。在发情期的雌性引起非性接受女性比男性更多的发声。 </li><li>男性表现出兴趣的女性（例如，嗅探，追逐，和/或安装）后，删除的女性和男性的发声记录为1-2分钟。 </li><li>男性如果不立即发声，简要地返回雌性大鼠男性的家庭笼刺激发声。 </li></ol> 2。声乐训练<ol><li>开始与Avisoft录像机的使用相同的配置，如图2所示的超声监测。 </li><li>在他家的笼子里的雄性大鼠下放置麦克风。 </li><li>男性的家庭笼放入一个雌性大鼠。如前所述，应在发情期的雌性大鼠，以最大限度地从男性的发声响应。 </li><li>一旦男性在女性的兴趣，消除女性。 </li><li>男性后，立即产生目标发声，同时奖励用笔点击并简要介绍了一瓶水或食物治疗。起初，任何50千赫的频率调制（图5B）调用的回报。随着训练的进行日益复杂的要求，如5至10呼叫快速连续的字符串的字符串，得到了加强。 </li><li>继续开展培训的会议，直到你给30增援。这通常需要5 - 10分钟之间。 </li></ol> 3。声学分析的准备工作<ol><li>始终归档原有的声音文件并进行编辑和工作副本的测量，分析，将需要过滤和清除每个文件的噪音。 </li><li> Avisoft SASLab Pro的使用批处理模式，高通滤波器在25 kHz分析，以消除不必要的噪音低于超声波发声的所有声音文件。 </li><li>为了使用SASLab Pro的自动检测功能，首先必须确定噪声阈值。要做到这一点，打开一个声音文件，并创建一个频谱使用图3所示的设置。 </li><li>自动参数测量显示，应根据图4所示的设置设置。 </li><li>查找那里有没有来电或噪音的频谱领域，这是背景噪音。 </li><li>使用“自动（单阈值）”选项，设置参数自动测量设置“对话框中的元素分离节只以上通过调整黑线以上频谱的功率谱窗口的背景噪音的门槛。在图4中，已设置的阈值-55分贝。 </li><li>在对话框的底部，设置“拒绝如果峰值幅度小于”的价值在上一步中找到的阈值低于1分贝。这两个值现在应该用于所有的声音分析的文件，假设所有记录的设置是相同的。 </li></ol> 4。发声鉴定<ol><li>标签使用上面设置的自动阈值的发声，首先要创建频谱图，与上面的设置。 </li><li>手动清除任何噪音，符合以下条件：1）被错误地作为一个发声，2）确定影响的开头和/或发声结束的歧视，和/或3）已被错误地鉴定为最高或在发声的频率最低（分别由上部和下部的红色线条，表示）。另外，发声标签的开始/停止时间可手动调整后，在下面的步骤4.5中创建永久性标签。然而，噪音影响的高频和低频测量，必须手动擦除。 </li><li>在某些情况下一个发声将无法从周围的噪音，如床上用品的运动过程中的声音，分离。在这种情况下，呼叫不能准确地分析和应删除。 </li><li>一旦所有的发声已经确定，永久删除声音文件的频谱选择删除部分“删除擦除波形的频谱部分... ...”从“工具”下拉菜单。如果不执行此命令前的频谱是封闭的，全部清除工作将丢失。 </li><li>元素分离部分的参数自动测量设置窗口中通过点击“编辑”按钮创建声音文件的永久标签。 </li><li>视觉和听觉审查每个标记的发声，以确定其类别：简单，频率调制（FM），或谐波。屁股IGN适当的标签，使用预先定义的文本模块。每个发声类的例子，如图5所示。 </li><li>关闭频谱图，保存声音文件，并继续下一个声音文件进行分析。 </li></ol> 5。声学测量<ol><li>在所有的声音文件发声后已经确定，使用批处理工具（工具：批处理）的所有文件的声学参数的自动测量。 </li><li>在批处理对话方块中，从下拉菜单中选择“自动参数测量”。 </li><li>选择“过程中所选文件夹中的所有文件”复选框，然后单击“文件夹”按钮，并选择声音文件保存的文件夹。 </li><li>点击“开始”，每个文件都将被自动分析，根据上次使用的参数自动测量设置对话方块设置。 </li><li>测量结果将被保存在一个文本文件，可以使用的DDE参数/日志文件设置命令定义。这个文本文件，然后可以导入到一个统计分析方案。 </li></ol> 6。代表结果： 13通常情况下，我们看到了以下的定量声学参数减少为特征的超声波发声质量的下降：受相关的生理变化与衰老和6 -羟基多巴胺帕金森病模型大鼠的超声波发声带宽，峰值频率，持续时间和强度。图6显示了在三个不同的时间点在PD模型大鼠的调频发声：基线后诱导的PD，发声训练后，。在PD状态下，呼叫显示在减少带宽，持续时间和强度。此外，频率调制，已成为不规则。 除了特定的声学参数的退化，我们也观察到在年龄和帕金森病大鼠的产生发声类型较少的整体复杂性。例如，在诱导的PD大鼠​​产生一个简单的电话和更少的频率调制呼吁更多。以下的发声训练，许多声学参数，如持续时间和强度，方法的基准水平，并增加复杂的通话记录的数量（图6）。老年大鼠类似的退化表现在他们的超声波发声的声学参数。目前，我们正在调查老年大鼠的声乐训练的效果。 <img src="/files/ftp_upload/2835/2835fig1.jpg" alt="图1"/> 图1：每节发声（各组大鼠的目标达到每星期）和发声率（均值和标准错误）都增加了一组两个年轻人（9月）和老为期6周的训练期间（ 32。 MO）菲舍尔344/Brown挪威F1大鼠。 <img src="/files/ftp_upload/2835/2835fig2.jpg" alt="图2"/> 图2代表50千赫的发声三类每个发声;（一）简单，（b）频率调制，和（C）谐波。 <img src="/files/ftp_upload/2835/2835fig3.jpg" alt="图3"/> 图3。调频代表从一个单一的大鼠（a）基线（B）诱导后PD，（c）在发声训练发声。

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动物实验是由美国国立卫生研究院实验动物护理和使用指南，实验动物，和动物福利法“的人文关怀和使用的公众健康服务政策所规定的准则和法规的规定执行。动物使用的协议是由美国威斯康星 - 麦迪逊大学医学和公共卫生学院的机构动物护理和使用委员会（IACUC）批准。

超声波发声在大鼠衰老和疾病过程，如帕金森病，似乎是脆弱的。这些超声波发声行为模型提供了一个发声功能，包括陪因疾病和衰老的生理变化的赤字。此外，在超声波发声的声学变化很容易测量和量化。此外，超声波发声可以修改通过行为干预。因此，大鼠超声发声提供一个有用的模型来研究疾病的影响，延缓衰老，和治疗干预行为及相关的神经生物学机制。 我们的声乐训练方法，使用条件刺激（点击），以积极的钢筋（水或食物治疗的奖励）。点击奖励是用来加强对日益复杂的发声。我们定义为那些与那些是“扁平化”和维护一个单一的频率（图5A）的“颤音”（频率调制）组件（图5B），复杂的叫声。这个频率调制分量是很容易识别其正弦的外观。而50千赫发声可能完全正弦（图5B），是很常见，遇到更加复杂的部分原因是这个频率调制元件以及频率或大跳跃或一个简单的平面组件组成的50 kHz的发声。这些更复杂的发声组件之间的不到10毫秒的短暂休息，但被认为是一个单一的发声。虽然这些发声进一步的详细子分类可能脱机，14分类的所有50 kHz的发声包含复杂的调制频率的组件，以减少需要在线主观分类和减少钢筋变异。在你自己的实验室，它是非常重要的数据是如何分类和分析，根据研究设计，研究的目的是适当的选择。 使用此协议时，有很多因素要考虑。首先，尽管遗传同质性和程序的一致性，老鼠有一个培训的变量响应。有些老鼠自然发声，5比别人更多的还是暴露比女性更多的食品或水奖励的动机。我们对这种变化帐户的方式之一是通过检查个别大鼠基线发声后病变和培训后的发声的声学参数的变化。虽然没有在本议定书的讨论，我们也有行为的分析，以量化的女性，如延迟装载的兴趣。此外，根据个人老鼠的年龄和活动水平，钢丝上方可能需要在培训过程中，以防止逃逸的家庭笼上。尽管个体差异，我们已经成功地用于对多种菌株和年龄相对每一个人大鼠的基线，以改善发声本议定书。 老鼠敏感环境的变化，如气味，温度和人员，看似很小的。应作出努力，以避免香味，如乳液，护发产品和香水，个人护理产品。试图训练之前，个人必须舒适的交互和处理大鼠。此外，在人员或环境的任何不一致可能影响老鼠的发声响应。 由于老鼠是夜间活动的，他们应该保持在逆光周期，以便培训，在黑暗的周期部分地方，当他们是最活跃的。我们照亮我们的培训室红灯。此外，如果在训练过程中使用水的奖励，老鼠应该有水的准入限制。我们的老鼠是培训会议之前21小时限制的水。三个小时与水接触的是由我们的机构动物护理和使用委员会限制最少的一段时间内，使水在我们的行为研究有用的增援，没有提出实质性的妥协动物健康或福祉。老鼠在随机时间（10分钟至30分钟）训练后获得水和随后的三个小时内有其光周期的黑暗部分自由饮水。脱水迹象，应每天检查大鼠和称重，隔日，以确保有没有显着性（大于10％）的减肥。 A的食物限制是没有必要作为奖励，如果使用的食品治疗。黄柏具体大鼠市售，但我们已经找到了一个含糖谷物的小片，是一种有效的奖励。 如上所述，在发情期的雌性大鼠引起非性接受女性比男性更高的发声响应。通过维持大量的女性在殖民地，有一个高的可能性，至少有一名女将会在任何一天的发情期。然而，发情期也可以将药理学诱导雌性大鼠皮下注射0.1毫升无菌麻油48小时10微克β-雌二醇前培训，0.1毫升无菌麻油训练前4小时500微克的孕激素。此外，必须有性经验是天真的雌性大鼠的雄性大鼠前声乐训练。这是通过放置一个雌性大鼠，将参展性接受男性的家庭笼，直到男性的坐骑和射精的迹象（疾飞，前凸，耳摆动）。男性可能无法装入前几届会议期间，通过嗅探和/或追逐笼子周围的女性，女性可能被拆除后的男性显示在女性的兴趣。性经历是每天一次，为期两周。这段时间内还提供了一个机会，老鼠habituate实验者（S）和录音室。 这种方法的一个局限是主观确定的发声类型。此外，这一因素限制了数据收集的速度，每次调用必须是独立的检查和手动分类。相关，这是谁一直在发声鉴定训练的有经验的评价者的重要性。区域内和跨评价者的可靠性，应在自己的实验室评估。 总之，在啮齿类动物中的超声波发声使用在许多不同的模型来研究脑-行为之间的关系，包括情绪状态，奖励和上瘾，和疾病状态， 如自闭症，6,14-16这种新颖的方法是一种研究的内在机制，有针对性的治疗老化和帕金森病的声音出现赤字的声乐训练。这种方法已被应用到其他疾病模型影响通讯和语音的潜力。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th></tr><tr><td>冷凝器超声麦克风CM16/CMPA </td><td> Avisoft Bioacoustics </td><td> 40011 </td></tr><tr><td> UltraSoundGate 116Hb </td><td> Avisoft Bioacoustics </td><td> 41161 </td></tr><tr><td> Avisoft的记录</td><td> Avisoft Bioacoustics </td><td> 10201 </td></tr><tr><td> Avisoft - SASLab PRO </td><td> Avisoft Bioacoustics </td><td> 10101 </td></tr></tbody></table>

targeted training of ultrasonic vocalizations in aged and parkinsonian rats

语音赤字是两种帕金森病（PD）和老化的常见并发症，它们可以显着减少的生活质量，影响交际能力的培养1，2有针对性的培训（语音/语音治疗）可以提高特定语音赤字，3，4虽然。行为干预的基本机制没有很好地理解。语音赤字和治疗系统的调查应考虑的因素很多，在人类，如年龄，家庭环境，年龄疾病发病后，疾病严重程度，和药物都难以控制。这里介绍的方法使用的发声的动物模型，允许基本感觉机制的变化有针对性的语音训练如何系统的研究。超声波的记录和分析，在本议定书中所列的程序，适用于任何啮齿类动物超声波发声的调查。 啮齿类动物的超声波发声正在成为一个有价值的模型，进行调查的行为的神经基础。5-8啮齿类动物和人类发声的价值进行符号，并通过修改egressive气流与喉收缩产生9，10因此，啮齿类动物发声可能是一个有用的模型来研究在感觉方面的声音赤字。此外，大鼠模型，使我们能够从有针对性的培训赤字研究恢复的神经生物学基础。 模型的PD我们使用长埃文斯大鼠（Charles River实验室国际公司），并诱发帕金森病的6 - 羟多巴胺（6 - OHDA）7微克到内侧前脑束，这会导致中度至严重变性突触前单方面输液纹状体神经元（详见Ciucci，2010）11，12为我们的衰老模型，我们使用的菲舍尔344/Brown挪威F1（国家老龄问题研究所）。 我们的主要方法是征求发声，揭露性经验的雄性大鼠性接受雌性大鼠。当男性对女性感兴趣，女性和男性继续发声。通过奖励复杂发声的食物或水，无论是复杂的发声和发声率可以增加（图1）。 超声波高于男性的家庭笼上的麦克风记录的发声。录制开始后，雌性大鼠被删除孤立的男性调用。发声可以查看实时培训或记录，并进行离线分析。通过记录和声学分析声乐训练之前和之后的发声，疾病和恢复训练的正常功能的影响进行评估。这种模式也让我们的大脑和神经肌肉系统的变化与所观察到的行为（声乐）改善。

Watch this Scientific Journal Video about Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats at JoVE.com

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

语音障碍衰弱老化和帕金森病。这些条件的影响，老鼠的超声波发声，也可以被用来研究这些语音障碍，他们的神经基础，和功能的恢复与行为干预的性质。

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 ...

targeted-training-ultrasonic-vocalizations-aged-parkinsonian

Research

JoVE Journal

Neuroscience

19.6K 次观看.  University of Wisconsin. 语音障碍衰弱老化和帕金森病。这些条件的影响，老鼠的超声波发声，也可以被用来研究这些语音障碍，他们的神经基础，和功能的恢复与行为干预的性质。

视频: Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

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

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

在大鼠的药物自我管理的评估超声发声

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

科研

神经科学

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

运动学与地面反作用力的测定：一个示范量化青年，中年和老年大鼠的运动能力能力

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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 relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

小鼠嗅觉学习和鉴别的客观和可重复性试验

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髓损伤 wistar 大鼠跑步机的作业训练

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Measuring Statistical Learning Across Modalities and Domains in School-Aged Children Via an Online Platform and Neuroimaging Techniques

Statistical learning, a fundamental skill to extract regularities in the environment, is often considered a core supporting mechanism of the first language development. While many studies of statistical learning are conducted within a single domain or modality, recent evidence suggests that this skill may differ based on the context in which the stimuli are presented. In addition, few studies investigate learning as it unfolds in real-time, rather focusing on the outcome of learning. In this protocol, we describe an approach for identifying the cognitive and neural basis of statistical learning, within an individual, across domains (linguistic vs. non-linguistic) and sensory modalities (visual and auditory). The tasks are designed to cast as little cognitive demand as possible on participants, making it ideal for young school-aged children and special populations. The web-based nature of the behavioral tasks offers a unique opportunity for us to reach more representative populations nationwide, to estimate effect sizes with greater precision, and to contribute to open and reproducible research. The neural measures provided by the functional magnetic resonance imaging (fMRI) task can inform researchers about the neural mechanisms engaged during statistical learning, and how these may differ across individuals on the basis of domain or modality. Finally, both tasks allow for the measurement of real-time learning, as changes in reaction time to a target stimulus is tracked across the exposure period. The main limitation of using this protocol relates to the hour-long duration of the experiment. Children might need to complete all four statistical learning tasks in multiple sittings. Therefore, the web-based platform is designed with this limitation in mind so that tasks may be disseminated individually. This methodology will allow users to investigate how the process of statistical learning unfolds across and within domains and modalities in children from different developmental backgrounds.

Presented here is a protocol introducing a set of child-friendly statistical learning tasks geared towards examining children&#8217;s learning of temporal statistical patterns across domains and sensory modalities. The developed tasks collect behavioral data using the web-based platform and task-based functional magnetic resonance imaging (fMRI) data for examining neural engagement during statistical learning.