作者要感谢总统的研究基金来自圣路易斯大学授予的WSN

1。人口笼的保养<ol><li>保持人口笼在25°C在12小时明暗周期。只要在对照组和实验组暴露在相同的照明条件，那么这种技术可以在一个标准的实验室设置进行。 </li><li>让女性在一夜之间产卵苹果汁琼脂平皿上。 </li><li>通过保持板与新蛋存放在25℃，24小时收集初孵幼虫。放置一小团酵母中的板的中心，以吸引孵出幼虫。 </li><li>收集已迁移到酵母膏苹果汁板中心第一龄幼虫。使用金​​属铲将其传送到一个​​新鲜的苹果汁板。可以添加额外的酵母膏，以确保有足够的食物，直到检测执行。葡萄汁板也可代替苹果汁板的使用。 </li><li>得晚2 次早3 龄升通过允许第一龄期年龄为40-48小时arvae。在此范围内的进料速度是恒定的13。年龄可以通过检查口钩被确认为有每个幼虫蜕皮明显的变化，这种结构。 </li><li>晚2 次早3 龄幼虫轻轻和充分洗涤苹果汁板与水和收集幼虫上的网状过滤器收集的行为分析。幼虫，然后转移到琼脂平板上。 </li><li>所有的分析都在使用平行对照组和实验动物进行。 </li></ol> 2。行为范式 - 步态<ol><li>将单份3 龄幼虫在2％琼脂基质在100mm组织培养皿中，并允许幼虫适应环境，持续30秒。幼虫用自己的cephalopharyngeal骨片（口钩）横跨琼脂基材表面推动自己的身体。这是在一个单独的板做，因为我t是更难以可视化体的收缩在酵母溶液作为动物几乎是相同的颜色作为酵母。 </li><li>观察并记录各后部到前部的移动在衬底上，为期1分钟。 N = 20，每基因型。不超过10个动物应每板来测定。 </li></ol> 3。行为范式 - 送料<ol><li>使用钝Inox公司＃5镊子，小心地从自发琼脂平板上转移3 龄幼虫，以琼脂填充板叠置用5毫升的2％活性面包酵母溶液的中心。确保解决方案是同质的酵母会沉淀一段时间。当在酵母溶液，幼虫会在很大程度上留在原地和饲料，便于观察的行为。的嘴钩收缩率直接相关，食物摄入14的数量。 </li><li>让幼虫来适应环境，持续30秒。 </li><li>观察并记录数量嘴钩收缩为一个周期为1分钟。 N = 20，每基因型。 </li></ol> 4。幼虫肠道夹层动脉瘤<ol><li>使在1x磷酸盐缓冲盐水（PBS）中，并置于有3孔点玻璃4％EM-级甲醛溶液固定。 </li><li>仔细剖析在外漂泊晚3 龄幼虫胆量使用1X PBS溶液3孔玻璃盘，确保每个腺胃保持不变的时间。用一个镊子，保持的后端，并与其他的，保持口腔钩子。按住后端不动，轻轻拉动嘴钩来访问胆量。删除任何相关组织（唾液腺，脑，脂肪体等 ），那么每个肠道转移到3井菜含有甲醛的修复。流浪3 龄幼虫的使用，因为在这个发展阶段，幼虫停止在准备pupariation觅食，腺胃被清除酵母。 </li> &lt;LI&gt;胆量​​孵育4℃过夜在一个不透明的组织培养箱。 </li><li>在从井中取出甲醛固定液，取出胃盲囊，并从腺胃夹肠〜150微米2，这样的预测可以清楚无阻碍观看。 </li><li>从孔中除去甲醛的修复和替换用1×PBT（1×PBS，0.1％无蛋白酶牛血清白蛋白，0.1％的Triton X-100）的缓冲溶液。在1x PBT彻底洗净胆6次10分钟。将组织样本上的机械旋转一边做洗。 </li><li>在10 -6 M的 5 -羟色胺，以提高血清素信号孵育在4℃下1小时。在1x PBT彻底洗净胆6次10分钟。以前的研究已经表明，外源性5-HT的浓度这不会影响在免疫分析中的神经元体系结构或静脉曲张密度和简单地提高了信号的信噪比15-16。 </li><li>孵育4＆＃176;过夜的抗血清素一抗（单克隆小鼠或多克隆兔提出提出）。在1x PBT彻底洗净胆6次10分钟。将组织样本上的机械旋转一边做洗。 </li><li>孵育在二次抗体4℃下进行90分钟（的Alexa Fluor 568羊抗鼠或抗兔IgG; 1:400稀释）。在1x PBT彻底洗净胆6次10分钟。将组织样本上的机械旋转一边做洗。 </li><li>孵育在4 10mM碳酸盐用于在机械转子10分钟钠，然后安装在4％的正丙基gallate/20毫碳酸钠，和视图下的荧光。碳酸钠是用于将样品转化为在封固介质中使用的缓冲液和pH值。 </li><li>捕捉免疫染色的组织样本图像在放大400倍进行分析。 </li></ol> 5。神经电路的分析<ol><li>轴突纤维进行定量（数量和静脉曲张的大小和支化的使用Neuroleucida和Neuroexplorer度）。然而，这也可以手动完成，或使用简单的轴突示踪（可以网上下载了一个免费的应用程序）。 </li><li>跟踪从大脑的各个纤维投射到腺胃和量化静脉曲张号码，枝和每单位长度大的静脉曲张的数目。轴突纤维从大脑中突出的捆绑到recurrens神经，不作分析访问，直到他们达到他们分开，其中腺胃和丛生。 </li></ol>

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我们什么都没有透露。

血清素stomatogastric电路，它在胚胎发育晚期出现的异常发育，将影响其成熟的功能。变化突起架构支配肠道可与电路的功能输出，这是进料速率（通过嘴钩收缩在酵母溶液测定）（图1）相关联。在果蝇中使用UAS-Gal4的二分体系使得可以特异性靶向上调或下调表达一个给定的转录物至特定组织的;改变一个给定的蛋白质的表达，可以精确地定量给予适当的工具。这种技术可以用来?...

果蝇是研究神经回路的发展，由于快速的生成时间，较低的实验成本，操纵和控制遗传和环境因素的能力是非常强大的模型系统。神经再生，神经元路径的发现和突触是人类和果蝇中保守，因此在创建，维护和修改神经回路机制是保守为好。 经典的神经递质，例如血清素（5 -羟色胺，或5-HT）可以作为生长因子通过它们的作用如同在成熟的神经电路1-3的信号分子前 以往的研究表明，扰动的5-HT水平胚胎发育过程中改变成熟神经元4的连通性。其他人已经表明5 -羟色胺对培养的神经元Helisoma异位应用抑制神经突生长以及突触5-7。 在 D中rosophila，发育的5-HT水平负相关静脉曲张数量和大小，以及aborization的程度，沿着突起从中枢神经系统8投射到前肠的长度。 血清素能神经传递已经显示调节摄食行为在不同的物种，包括果蝇 8-9。馈电电路在果蝇是可以作为一个模型关联功能的输出（馈送）与在轴突突起从大脑前肠的发展改变一个相对简单的电路。 Schoofs 等 。已经表明， 果蝇幼虫取食是由中枢模式发生器，影响肌肉组织10规范。而特定的肌肉解剖尚未完全了解，但已经表明，触角神经，上颌神经和前胸腺副神经负责参与肌肉目标摄食行为。涉及的无脊椎动物的摄食肌肉和神经解剖大多数数据是有限的丽蝇幼虫。 第二龄期幼虫的进料速率可以通过cephalopharyngeal骨片（口钩）的回缩进行评估，并且是可重复的和高吞吐量。该cephalopharyngeal板由中枢5-HT能神经元纤维通过额叶神经支配。腺胃，或前肠，是由血清素纤维（recurrens神经），该束状中肠，并负责前肠的收缩（ 图1）11-12支配。变化的轴突分枝和静脉曲张沿神经突长度的数量和大小，可以用免疫组织化学技术进行定量。在开发过程中操纵神经元的5-HT，无论是直接或间接地，可改变该供电电路的功能输出，其可以进行评估，并以变化的morpholo相关GY的突起结构。

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:217px;">Eclipse E-800 Microscope</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:217px;">Neuroleucida</td>
			<td style="width:175px;">MBF Biosciences</td>
			<td style="width:115px;">NL-15</td>
			<td style="width:251px;">Used to analyze gut fiber architecture, not necessary to have</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:217px;">Northern Eclipse</td>
			<td style="width:175px;">Empix Inc</td>
			<td style="width:115px;"></td>
			<td style="width:251px;">Imaging software</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:217px;">G-2E/C TRITC EX 528-553</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">96312</td>
			<td style="width:251px;">Filter for specific secondary antibody</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:217px;">N.A. 0.75; W.D. 0.72 mm; DIC Prism: 40xI, 40x I-C; Spring loaded</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">MRH00400</td>
			<td style="width:251px;">Objective used for imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:217px;">Simple Neurite Tracer</td>
			<td style="width:175px;">NIH Image J</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"><a href="http://fiji.sc/Simple_Neurite_Tracer">http://fiji.sc/Simple_Neurite_Tracer</a></td>
		</tr>
	</tbody>
</table>

functional analysis of the larval feeding circuit in drosophila

的血清素激活的供电电路在果蝇幼虫可用于该电路的开发过程中，调查至关重要的神经基质。使用该电路的功能输出，馈送，改变stomatogastric系统的神经细胞的体系结构可以被可视化。摄食行为可以通过观察口的钩子，它从大脑接收支配收缩的速率进行记录。自发行为作为一种生理控制觅食，因为幼虫用自己的嘴钩来遍历整个琼脂基质。改变摄食行为可以与突起的轴突架构相关联支配的内脏。采用免疫组织化学也能够可视化和定量这些变化。幼虫在行为范式操作不当可能会改变数据，因为他们是操纵非常敏感。适当的成像轴突架构的支配肠道是膨体的数量和尺寸的精确定量以及分支节点的程度是至关重要的。大多数电路的分析只允许对神经突的体系结构或行为效果的视觉效果，但是，这种模型允许一个相关联的电路与在神经元体系结构的损伤的功能性的输出。

血清素供电电路中的果蝇幼虫可以作为一个非常有效的模型，观察特定因素对神经系统发育的影响。通过定量进料速率，因此能够与它的功能性输出（图1）连接的馈电电路的轴突的结构。的运动功能测定法是用来作为一种生理控制的口钩的后退动作中，由于幼虫用他们的嘴钩穿过琼脂表面推动自己。应在控制和突变基因型之间的自发反应没有区别，如果突变只影响供电电路<str...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

供电电路在果蝇幼虫提供了一个简单但功能强大的模型，它允许改变喂养率与在stomatogastric神经回路的改变相关。这个电路是由该发送突起的钩口以及前肠中央血清素能神经元。

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

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Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

10.2K 次观看.  Saint Louis University School of Medicine. 供电电路在果蝇幼虫提供了一个简单但功能强大的模型，它允许改变喂养率与在stomatogastric神经回路的改变相关。这个电路是由该发送突起的钩口以及前肠中央血清素能神经元。 

视频: Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

科研

神经科学

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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 ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...