This protocol follows any and all animal care and use guidelines and regulations set by IACUC at the University of Cincinnati. This work is funded by NIH/NINDS NS07722 and the Gardner Family Center for Parkinson&#8217;s Disease and Movement Disorders.

所有步骤的协议遵循动物护理和使用由辛辛那提大学的IACUC设定的准则和法规。 1，一般注意事项<ol><li>如果可能的话，在此期间在黑暗周期的活跃期测试小鼠。通常情况下，进行低光照下，并在至少1小时，检测到黑暗的循环。然而，如果测试在暗周期是不可行的，光周期期间执行所有三个测试。请记住，当老鼠在他们的睡眠周期被嗅探测试时间可能会减少。注意:当测试可以在睡眠周期中执行的老鼠可能会显示在埋颗粒，块，或香味的墨盒不感兴趣。 </li><li>为了减少外部嗅觉暗示的任何效果，穿碱基对手套在任何时候都贯穿每个试验，然后将清洁的一对手套在碱基对每只小鼠和每个试验。一般来说，如果有疑问，换上了一套手套的清洁。有一个盒子手套读随手可用，因为它们会经常发生变化。注:我们用丁腈手套所有动物处理。 </li><li>清洁测试材料和受试者用消毒之间笼/消毒剂也正是在这种电池的试验的一个重要步骤，以减少潜在的疾病传播，以及除去不需要的嗅觉暗示。 </li></ol> 2，埋颗粒测试<ol><li>在测试前至少两天，记录每只小鼠的体重，然后食物限制到90％的体重。在测试之前和期间，食物限制，给每只小鼠1-2件粒料的测试过程中使用（ 例如，一块甜化谷物的）。不要跳过这一步，因为老鼠是恐新食品和测试过程中不得搜查的颗粒，如果他们都没有吃小球之前。 </li><li>在所有的测试天，通过将笼子的考场，没有一瓶水或器B的，观察者小鼠1小时前测试英寸房子他们在笼子只有一个过滤器顶盖。 </li><li>在习惯化，填补了清洁鼠笼〜3个厘米高有干净的被褥使某些被褥均匀地分布在整个笼子。 5分钟设置一个计时器。 </li><li>继1小时的习惯，埋在下面的床上用品1加糖谷物颗粒0.5厘米所以它是不可见的。 </li><li>从家笼中取出测试鼠标，将其放置在试验网箱的中心，将过滤器顶盖上的笼子里，开始计时。 </li><li>停止定时器，当鼠标揭示沉淀，并开始吃它。注意，露出埋丸所需的时间。 </li><li>如果鼠标没有发现在5分钟内颗粒，停止审判，并记下该鼠标的得分为300秒。将沉淀物以这样的鼠标可以访问它的寝具的顶部，并允许小鼠吃的粒料。 </li><li>下面的试验中，返回鼠标的家笼。 </li><li>清空从测试笼的床上用品和清洁的C年龄与动物房清洗溶液。 </li><li>重复步骤2.4至2.9为每个滑鼠，在每次鼠标后更换手套。 </li><li>毕竟小鼠进行试验，给他们足够的食物来维持在90％的体重。 </li><li>执行第2-5天以同样的方式测试作为测试第1天有一个例外;埋葬沉淀在笼子里的每个试验不同的位置。 </li><li>在测试的第6天，进行表面颗粒审判。 </li><li>对于表面颗粒试验，遵循相同的程序测试第1天但不是在床上用品将它放在被褥上面埋葬的沉淀。记录时间为鼠标找到并开始进食沉淀。 </li></ol> 3，测试座<ol><li>个别家鼠在清洁笼子里，并放置5块笼（标记为AE），每个网箱设置改变之前的手套。 </li><li>随后进行测试24小时。移动测试小鼠的试验区，取出一瓶水和馈线箱，然后将人升从笼子里有5个街区还有少数床上用品装入塑料袋标有动物的身份。放置在密封的塑料袋上鼠标的笼子的顶部。 </li><li>的，观察者动物在其没有水瓶，给料机仓笼，并阻止1小时前测试。 </li><li>更改每笼之间的手套，这样的气味是不是动物/块之间交换。 </li><li>排队对方旁边的笼子鼠标在桌子上，每个网箱之间至少有10厘米。放置一台摄像机，使笼（不笼的长边）的前面是明确的说法。 </li><li>设置定时器30秒，标记有一个重要的实验细节（日期，鼠标的ID，审判等 ）的记忆卡。录像带的标签为2-3秒。 </li><li>从塑料袋中与鼠标的标识删除块广告并将其放置在笼子的中间，使他们清楚地摄像机上可见。确保有〜1厘米Ø每个块的F之间的空间。将过滤器顶部回到笼子里。启动定时器和录像带，持续30秒。 </li><li> 30秒后，停​​止录像并从笼子块，并把它们放回塑料袋。 </li><li>更换手套，然后转移到下一个鼠标，重复用于第一试验同样地进行。做到这一点，共计6项研究对每只小鼠确保有中〜5分钟的试验间隔。 </li><li>在第 7 次试验，遵循相同的程序为试验1-6除了没有把块D从鼠标本身的笼子从另一个老鼠的笼子里添加E座让鼠标从接触到块A，B和C的从另一个老鼠的笼子自己的家笼及E座。注意，在实验室中书的方框E各小鼠暴露于和备用其中方框E被放置在每只小鼠的笼子（ 例如 ABCE或AEBC）。以E座从鼠标同性而不是从原始cagemate。六deotape 30秒。 </li><li>继7项试验，动物回归到一个新的干净的笼子与原cagemates。用于测试用中性清洁剂和水的清洁块，晾干在后面的测试中使用。 </li></ol> 4，习惯化/ Dishabituation <ol><li>以类似的方式，以块测试执行习惯/ dishabituation测试，但代替使用具有鼠标的气味块，使用保持棉花香味的不同对提取物的组织盒。 </li><li>按照步骤3.1-3.4使用一个单一的，无味的组织盒，而不是块。 </li><li> ，观察者的小鼠在试验室。 </li><li>在一个单独的房间准备香味组织墨盒。将小棉球放入干净的墨盒，然后加入5微升提取物（ 如杏仁萃取物）成绵。捕捉墨盒顶部在棉花和发生在专用的盒子或袋子标记与香味。 </li><li>做同样的程序F或第二的气味（ 如八角提取物）。改变处理不同气味的手套。确保棉花球，在组织盒完全包封。注:裸露的棉花球纤维可导致动物在棉花球嵌套的目的，而不是气味本身的兴趣。 </li><li>然后，按照步骤3.6-3.10使用一种带香味的组织盒的第6项试验，第二香味组织墨盒第七审判。 </li></ol>录像带的五分析<ol><li>对于该块和习惯/ dishabituation测试，有一个评估者盲目的实验条件下测量了嗅探每个块，用嗅探定义为与该块鼻腔接触的时间。如果需要的话，测量的其他行为，如时间到试验7和总活性各试验期间（沿长度和宽度的家笼所作运动的次数）在接近新的嵌段（E）。分数嗅花的时间ð时间从用秒表录像带接近。 </li><li>同样，对于习惯/ dishabituation测试，有一个评估者盲目的实验条件下测嗅花的墨盒，时间接近墨盒，自主​​活动的时间。 </li></ol> 6，统计<ol><li>分析采用非参数统计，因为等待时间，找到小球得分往往表现出差异的异质埋颗粒测试。平均等待时间以找到在整个3-5天的沉淀，然后用曼 - 惠特尼U检验的小鼠组之间的比较。类似地，对于测试的表面沉淀部分，分析延迟使用曼 - 惠特尼U检验比较基因型发现沉淀。 </li><li>对于该块试验，分析试验从7（引进新的香味块）中的数据。通常情况下，采用非参数的分析来解释方差的异质性。为了分析在时间差使用了嗅探日ê小说块以及时间接近块不同实验组，用曼 - 惠特尼U检验。使用Wilcoxon符号秩检验比较花时间嗅探小说块花费的时间嗅着熟悉的块。 </li><li>对于习惯/ dishabituation试验，进行参数分析了试验1,6和7，类似于以前的研究20。如果小鼠表现出几乎没有嗅探，使用非参数的分析。 </li></ol>

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We have no conflicts of interest.

每个在此研究中所描述的测试测量嗅觉功能的小鼠的不同方面。埋颗粒测试测量嗅觉的粮食动机方面，测试的饥饿（食物的限制，而不是禁食）来检测可口一块甜麦片埋在被褥能力的小鼠。块试验测量更嗅觉功能的社交方面，检测小鼠自身气味和的同种之间进行区分的能力。习惯化/ dishabituation试验评估了小鼠的熟悉和新颖的，无害的气味来区分的能力。在鉴定一个新的模型，因为食物中的动机或恐惧异常有助于在突变小鼠中观察到与对照组相比差异使用多个测试是非常重要的。例如，如果仅在块执行测试，并降低嗅探已另一个鼠标的气味被观察到的块，也不清楚是否有olfact储器缺陷或增强的恐惧反应，可能导致回避该块的同种。当多个测试执行和缺陷在所有试验中观察到，它强烈支持的嗅觉障碍的解释。但是，如果差别只在一个测试，但没有其他人看到那么有可能是一个更微妙的嗅觉障碍，但是这将是必不可少的，以排除其他的解释（ 即增强恐惧或减少食物的动机）。测试嗅觉功能的小鼠时，要记住其他重要因素，包括小鼠的背景和应变性。不同遗传背景的品系（ 即，C57BL / 6，DBA， 等等 ）可以具有对小鼠行为产生深远的影响，因此，建议在进行整个实验前，以适应在同一背景的野生型小鼠的每个协议。此外，还建议在雄性和雌性小鼠分别测试彼此因为男性的嗅觉系统是高LY到女性发情和嗅觉检测的发情雌性敏感可能干扰在所有三个测试中的表现。 有些步骤是绝对关键的小鼠嗅觉测试时要遵循的。要弄清实验者被引入到动物的气味是重要的。戴手套的程序是动物之间手套必不可少的，频繁的更换是必要的。它始终是为实验者不穿古龙水或香水的嗅觉测试天一个很好的做法。而该过程的某些部分是不灵活的，也有可以进行修改和改编，而不降低测试的有效性或灵敏度等部位。例如，习惯化试验的数目可以增加或减少取决于小鼠的菌株进行测试，以及如何快速地它们，观察者对刺激的反应。这些测试的主要限制是，它们都被驱动通过某种形式的动力，无论是食品或社会，使得难以完全排除异常动机作为解释当改变的响应变化。这可以通过测试和时间接近的刺激中分析的其他参数，如活性被最小化。 一旦掌握了这些测试可以很容易地用于研究的神经退行性疾病的新的小鼠模型的表型。此外，具有最高功率的测试可以包括在临床前研究中测试的潜在疗法（对于一个例子见参考文献25）。

嗅觉障碍是与一些神经变性疾病，包括帕金森氏病（PD），阿尔茨海默氏病和亨廷顿氏病1。在PD，嗅觉障碍包括在气味识别，检测和识别缺陷和被发现在高达的患者2-5 70-95％。这些缺陷可以通过长达4年之前PD的大是大非运动症状，表明嗅觉障碍可能预示6-10帕金森的早期阶段。在帕金森病早期出现嗅觉赤字已经导致嗅觉障碍所涉及的基本机制产生了浓厚的兴趣和。在啮齿类动物的临床前研究，嗅觉障碍可能是一个敏感的测量结果来预测新的治疗策略的治疗潜力。 许多测试已经设计并广泛用于帕金森病的啮齿动物模型和TES表征感觉障碍吨的新的治疗方法11-15的治疗潜力。尽管嗅觉赤字是有据可查的帕金森病，嗅觉功能尚未经常在很多车型进行测量。这种观点正在发生变化，虽然与遗传形式的帕金森病的发现和比较公认的观点，即帕金森病是一种全身性疾病影响的不仅仅是感觉功能。目前，有在帕金森病和其他神经变性疾病的遗传小鼠模型大量的研究，现在包括嗅觉的分析中表征16-24。给定的嗅觉障碍在神经退行性疾病的越来越大的兴趣，我们试图组装的，可用于临床前研究以表征神经变性的新模型，以及测试潜在的疾病修饰治疗嗅觉测试的电池。在本研究中所描述的试验中采用了两个特征和临床前研究18,25。 测试极力推ighted在这项研究中已被证明是在帕金森病（18）的频繁使用的α-突触核蛋白过表达的小鼠模型中检测嗅觉障碍敏感。它们包括掩埋沉淀试验26,27，块试验23,24,28,29的改编版本，并且习惯/ dishabituation测试30。需要注意的是有位在小鼠嗅觉功能的敏感措施在本研究中所描述的测试的几个适应是非常重要的，那些在本研究强调是测试我们的实验室拥有最经验和常规使用的。

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olfactory assays for mouse models of neurodegenerative disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

掩埋丸，块，并习惯/ dishabituation测试都是嗅觉小鼠的高度有利的评价。使用这些试验中，我们发现在嗅觉在帕金森氏病的多个基因小鼠模型，包括α-突触核蛋白过表达（大鼠Thy1-ASYN）18和Atp13a2敲除小鼠显著变化。这两种大鼠Thy1-ASYN和Atp13a2基因敲除小鼠需要较长时间才能找到埋藏颗粒比对照组18。 图从野生型和Atp13a2基因敲除小鼠收集1-3显示的数据。在埋入沉淀试验，Atp13a2敲除小鼠显示增加的延迟，以找到掩埋粒料相比野生型对照小鼠（ 图1）。在块试验都野生型和Atp13a2敲除小鼠嗅探从另一小鼠的笼子的新颖块相比，从自己家笼（ 图2）块。在习惯/ dishabituation测试野生型小鼠表现出习惯一个气味和<html>然后，当增加了一种新的气味引入（ 图3）嗅。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51804/51804fig1highres.jpg" width="500" /> 图1。埋颗粒测试。延迟发现沉淀在野生型（N = 10）和Atp13a2 KO（N = 14），在20-27米以上。 * P &lt;0.05，曼 - 惠特尼U。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51804/51804fig2highres.jpg" width="500" /> 图2块测试时间嗅探小说块（五）在该块试验中野生型组（n = 10）和Atp13a2 KO（N = 12）的雌性小鼠20 - 27米岁。 ΔΔ表示P &lt;0.01相对于块A，B和C从同一基因型。采用Mann-Whitney U。 "SRC ="/文件/ ftp_upload / 51804 / 51804fig3highres.jpg"WIDTH ="500"/&gt; 图3:习惯化/ dishabituation测试，平均时间在嗅野生型小鼠香味盒（9米的年龄，N = 7），在6个试验，并引进墨盒与一种新型香味审判7。

Watch this Scientific Journal Video about Olfactory Assays for Mouse Models of Neurodegenerative Disease at JoVE.com

Olfactory Assays for Mouse Models of Neurodegenerative Disease

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

嗅觉测定的神经退行性疾病的小鼠模型

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

olfactory-assays-for-mouse-models-of-neurodegenerative-disease

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21.8K 次观看.  University of Cincinnati. Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

视频: Olfactory Assays for Mouse Models of Neurodegenerative Disease

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

脑室周围白质软化的小鼠模型

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

科研

神经科学

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化:隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.