这项研究是由赠款没有。 0723341（KGL）从国家科学基金会。我们也感谢夏皮罗本科生研究奖学金计划和伦道夫 - 梅肯学院心理学系提供的捐款。最后，我们对此表示赞赏克雷格金斯利的协作贡献，在这项研究和Rzucidlo阿曼达的帮助，在此视频文章准备的R - MC实验室。

1。动物模型的识别<ol><li>加州的鹿鼠（Peromyscus californicus）是一个理想的模式，探索父亲的反应。正如您所看到的，这款鼠标新郎的幼崽，就像妈妈做的 - 他们甚至超过他们蹲下身，所以，它看起来像仔兔哺乳期。加州小鼠的行为相比，同属一个物种，它在幼崽表现出的兴趣不大，往往试图逃跑，甚至攻击他们共同的鹿鼠 （Peromyscus maniculatus）。 （参见图1这些相互作​​用的物种与幼仔的照片。） </li><li>一旦该物种的模式已被确定，则需要建立各团体。在这项研究中，亲生父亲，没有育儿经验的处女，并使用有限的小狗曝光的这两个物种（PUP暴露或寄养的父亲）的处女，所以，父亲的倾向和后天的特点，可以进行评估。在这项研究中，我们也暴露了所有群体，一个玩具鼠标小狗，以保证社会互动具体到一个小狗，而不是放置在笼子里的任何一个代表响应（参见图2。） </li><li>调查神经生物学因素之前，重要的是，以确认所选择的物种明显的差异表现在利益的行为。在这项研究中，男性为五分钟，一个父亲的行为ethogram（见ethogram例如表1）小狗放在，是用来得分的行为，如延迟接近小狗和接触的时间花在一个笼子里小狗。如果积极的反应，小狗立即删除。所以可以随后进行了仔细行为分析，这些会议往往录像。 </li></ol> 2。准备脑组织<ol><li>以下的每个鼠标的标准灌注（协议附后），被删除的大脑，使他们能够在感兴趣的特定区域（显示大脑）切片。 </li><li>一个标准的老鼠大脑图谱（查看图集）用于定位于下丘脑室旁核，已知的细胞产生的神经肽在这项研究中的兴趣丰富的地区 - 加压素（AVP）。 </li><li>大脑，然后封锁和冻结夹头安装，使组织的切片机切片前，将冻结。大脑是在修剪模式，切片，直到特定的地标被确定，那么在这种情况下，30微米，在特定的大脑厚度是用于分析的脑切片。 </li><li>脑切片小心放置在充满磷酸盐缓冲液（PBS）的孔板;（PBS附加议定书“;请注意，这是唯一的一个标准的免疫细胞化学协议的一个例子）。 </li></ol> 3。免疫细胞化学<ol><li>纵观这一过程的各个步骤（见附件协议），重要的是有准确的移液技术，这种技术（显示学生吹打），化学品和其他重要成分，以保证精确的测量。 </li><li>在这个过程中的第一步是洗脑筋，其中包括更换孔板的三至五倍的PBS之间的每次清洗孔板放置在一个10分钟的摇杆（显示学生更换PBS和名次上的摇滚乐队孔板） 。 </li><li>大脑切片，然后接触到的主要抗体的解决方案，并保存在4 ° C的摇杆（展示过程）过夜。 </li><li>另一个洗之后，大脑切片暴露到二级抗体在室温下摇臂一小时，然后清洗一遍。 </li><li> Avitin -生物素的复杂的神经化学物质的阳性细胞的可视化解决方案，准备下一步的大脑暴露。 </li><li>对于最终的可视化步骤，大脑暴露民建联。这是一个危险品，应始终谨慎处理。正如在这里看到，大脑切片开始之前得到你的眼睛暗（展示过程）。 </li><li>民建联曝光后，大脑经过清洗的最后一个系列，然后小心地放在subbed显微镜载玻片和干通宵（显示幻灯片上的脑片的位置; subbed幻灯片协议附件）。 </li><li>幻灯片，然后通过一系列蒸馏水和酒精清洗清除，终于被淹没在Citrosolv前coverslipped和储存保管（显示这一过程的各个方面;清算过程中看到的附件）slidebox。 </li></ol> 4。 Neuroquantification <ol><li>幻灯片已经干涸后，他们可以使用专门neuroquantification软件进行评估。这里Bioquant软件是用来量化在老鼠大脑的下丘脑室旁核加压素阳性细胞和纤维（显示屏幕上的软件... ...和显微镜）。 </li><li>感兴趣的特定区域使用的测量选项确定软件建立neuroquantification视野。重要的是一致的视野大小是每个动物的量化（显示学生这样做）。 </li><li>在这里，深染加压素免疫反应阳性胞体和纤维是显而易见的。因为它是难以计数或跟踪这个组织，这个软件的特殊功能使用光阈值，以确定阳性染色的组织利益的指定区域内的总金额。这个阈值告诉我们有多少指定的区域中加压素阳性组织（显示在计算机屏幕上的数据值）。 </li></ol> 5。行为分析<ol><li>如果超过一名观察员将得分录影带，重要的是要建立跨评价者的可靠性，以确保观察员得分以一致的方式行为。对于实际得分会议，行为评分表应准备允许轻松得分的行为观察会议期间（例如得分试算表，请参阅表2）。 </li><li>行为，偶发的反应并不快，一个microsequencing分析，可以揭示有关的具体回应进展的微妙之处。例如，修饰行为组成一个链的反应速度非常快。虽然一个选项是简单地记录修饰的存在和持续时间，另一个是文件伴随这一反应的事件链。使用这种microsequencing软件，观察员得分的特定的行为每秒钟软件（显示软件和视频）的提示，存在。 </li><li>数据收集，感兴趣的参数确定和分析适当的行为分数。例如可能接触的时间花在与小狗，或在疏导序列中断（每个动物输入的数据表显示与传播计算机）的总时间。 </li><li>一旦行为取得重要的是，以确认这两个品种表现为行为的研究兴趣不同的应对策略。加州鹿鼠在这种情况下，应表现出更多的父系比普通的鹿鼠的行为。此外，各种行为的措施，可与大脑的措施，以获得一个更明智的有关影响。 </li></ol> 6。代表性的成果： <ol><li>来验证模型，数据清楚地表明，这两个品种的表现不同方面利益的行为。这里你可以看到，P。 californicus男性花费更多的时间疏导和幼崽，两个父亲的反应的特点（见图4）下蹲。 </li><li>为了确定利益neurobiologial变量是重要的，在父亲的反应，加压素（AVP）的积极组织几个相关的大脑区域进行了量化，可以看出 ，父亲的体育在这些脑区的几个 californicus动物免疫阳性组织。 （见图5）。 </li><li>在相关的研究使用microsequencing分析，推测，父亲的加州小鼠会表现出比他们的处女的焦虑少，因此，当暴露在捕食者的气味，父亲展出更少的中断，比处女动物美容序列（见图6）。 </li></ol><img alt="图1" src="/files/ftp_upload/3173/3173fig1.jpg" /> 图1：（一）男加利福尼亚州的小鼠表现出对同种外来小狗父亲的反应。 （二）男鹿鼠标表现出谨慎的态度（拉伸出席）和避免存在一个同种外来小狗的反应。注意：在这些社会互动评估，如果一个男性的展品对小狗积极的反应，实验者立即打笼顶部分散男性。此时会话终止，小狗被删除，任何伤口进行检查，并回到母亲。在我们的实验室中，这种情况很少观察与体育californicus男性，但偶尔观察与体育maniculatus;密切观察，并立即进行干预，但是，防止发生动物伤害。 <img alt="图2" src="/files/ftp_upload/3173/3173fig2.jpg" /> 图2：一个玩具鼠标交互加州鼠标，大多数试图对这些刺激咀嚼。 <img alt="图3" src="/files/ftp_upload/3173/3173fig3.jpg" /> 图3：研究的实验设计;有每个品种约6各组动物。 <img alt="图4" src="/files/ftp_upload/3173/3173fig4.jpg" /> 图4：图形的免疫细胞化学过程。 <img alt="图5" src="/files/ftp_upload/3173/3173fig5.jpg" /> 图图5在加利福尼亚州和鹿鼠的父系反应;父亲的反应（新郎，蹲下身，更快的方法）在加利福尼亚州小鼠观察。更舒展出席（应激反应），观察鹿鼠。 <img alt="图6" src="/files/ftp_upload/3173/3173fig6.jpg" /> 图6。加压素在这两个物种的不同脑区的免疫反应。加州的小鼠有更多的丘脑室旁核细胞和纤维的染色。 <img alt="图7" src="/files/ftp_upload/3173/3173fig7.jpg" /> 图7：父亲加州的小鼠表现出在自己的仪容序列比小狗暴露和维珍集团的捕食者气味的存在较少的中断。

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没有利益冲突的声明。

本文讨论三个基本组件被认为是行为神经科学调查的成功执行的关键：（1）一个具有代表性和有效的动物模型;（2）准确，灵敏的免疫细胞化学的协议;（3）大多数分析（观测和统计）双方的行为和大脑的数据。一个类别中的错误，肯定会危及整个研究的结果。因此，仔细选择合适的动物模型后，相当大的努力应该是指向进行试验的行为和组织学的程序，以确保该行为将被可靠地在研究中观察到，成功的大脑处理。 如前所述，使用比较物种找出一个特定的反应神经生物学机制是一种宝贵的的的方法的方法，因为这种技术并不需要基因工程或痛苦的手术操作。因此，这种方法的方法利用不足的威胁，更完整的动物。不过，使用动物的自然变化，加强了类似自然的环境纳入即使是在实验室内的动物。此外，如果实验室的研究可以扩展到的领域，进一步验证了物种的差异，在利益的行为的真实性，这一步建议。虽然比较的方法具有许多优点，限制是相关数据的性质，因此，应采用其他技术，如药理操纵利益的行为，进一步验证了有针对性的神经生物学系统的作用。 一旦实验者有关的行为，组织学和统计程序感到有信心，应采取使所有组动物的生活条件仍然不变，当然，实验操作除外。轻时间表，噪音水平，管理员，湿度水平和实验室中的气味，如变量的变化可能对动物的神经生物学反应有显著的影响。 在这篇文章中的主要抗体用于加压素的检测，但其他主要的抗体，可用于大量不同的神经化学物质的利益使用。如果实验者是一个新的抗体的工作，重要的是要进行滴定研究，以确定新抗体的最佳稀释。很多时候，太多的抗体（由供应商的建议），在组织太暗分化的信号;进一步，过度使用的抗体是非常昂贵的。 最后，如​​果不是一个单一的统计分析，可用于提供备用的意见和对数据的解释，他们应该利用。在这项研究中，用于提供一般线性模型，相关性，以及多维尺度分析的数据最翔实的意见。

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using a comparative species approach to investigate the neurobiology of paternal responses

行为神经科学的目标是，以确定潜在的神经生物学因素，具体的行为规范。利用动物模型来实现这一目标，许多方法论上的战略需要微创技术来操纵的利益的行为的力度（例如，病变的方法，药理操纵，微透析技术，基因工程动物模型）。利用比较物种的方法，使研究人员能够利用自然产生的响应现有的战略密切相关的物种差异的优势。在我们的实验室，我们使用两个Peromyscus属的物种，在父亲的反应有所不同。男加利福尼亚州鹿鼠（Peromyscus californicus）展品为女性，而其表弟同父母的反应，常见的鹿鼠（Peromyscus maniculatus）展品几乎没有培育/父母的反应，在幼崽的存在。在这篇文章中的具体利益是由父系加州鹿鼠标展出affiliative社会反应相关的神经生物学因素的探索。因为行为神经科学的方法是多方面的，以下关键组件的研究将简要讨论：这种类型的研究适当的物种鉴定;行为分析的数据收集，准备和大脑切片;免疫细胞化学法对涉及的基本步骤加压素免疫反应的量化，量化脑组织的神经影像软件的使用;使用一个microsequencing视频分析，行为得分，最后，适当的统计分析，提供研究结果的最明智的解释。

Watch this Scientific Journal Video about Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses at JoVE.com

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

比较种方法，使行为神经科学家探索与被视为一个特定的动物模型的特点特定的行为有关的各种神经生物学因素。利用自然发生的行为密切相关的物种之间的差异，这种技术不需要侵入性技术来操纵行为的表达。

使用方法比较物种调查父亲的反应神经生物学

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

using-comparative-species-approach-to-investigate-neurobiology

Research

JoVE Journal

Neuroscience

12.5K 次观看.  Randolph-Macon College. 比较种方法，使行为神经科学家探索与被视为一个特定的动物模型的特点特定的行为有关的各种神经生物学因素。利用自然发生的行为密切相关的物种之间的差异，这种技术不需要侵入性技术来操纵行为的表达。

视频: Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

小龙虾的反思记录行为反应

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

，测量主观价值风险和不明确的选项，使用实验经济学和功能磁共振成像的方法

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the &quot;Loser&quot; Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

一种新的方法，省去了处理的研究侵略和“失败者”的影响<em&gt;果蝇</em

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...