我们要感谢奎因皮亚克大学的凯勒和玛吉实验室的所有成员，提供有用的建议。特别是，我们要感谢Barron L. Lincoln II在凯勒实验室内开发这种损伤测定。我们还要感谢Quinnipiac大学艺术与科学学院授予LC Keller的赠款。

1.试剂和设备的制备<ol><li>制备1X夹层缓冲液（70 mM的氯化钠，5mM的KCl，0.02 mM的氯化钙 ，20mM的MgCl 2的，10mM的碳酸氢钠 ，115毫蔗糖，5mM的海藻糖，5mM的HEPES; pH 7.2）中。 </li><li>准备1克磷酸盐缓冲盐水（PBS）。 </li><li>使用含有0.01％Triton X-100的1x PBS制备1x PBT。 </li><li>准备果汁琼脂平板。 14简要地说，在700毫升混合的H 2 O和高压釜30克琼脂。将0.5g对羟基苯甲酸甲酯溶于10mL乙醇中，并将溶液加入300 mL果汁浓缩物（葡萄或苹果）中。将浓缩物混合到高压灭菌的琼脂溶液中，倒入10 mm x 35 mm培养皿的盖子中。 注意:葡萄琼脂功率预混还可以从各种公司购买（参见材料表 ）。 </li><li>获得大小5镊子，允许一个机械伤害幼虫。 </li><li>使用标准果蝇 CO 2 </s基于麻醉的麻醉装置。 </li></ol> 2.选择果蝇幼虫<ol><li>拿一瓶或一瓶果汁，含有在25°C培养的流浪三龄幼虫。 </li><li>根据外观选择十只个体第二龄或第三龄幼虫。三龄幼虫可以通过前分枝螺旋体的外观鉴定，而二龄幼虫较小，具有更多的杆状前叶。 注意:幼稚园阶段也可以按时间确定。在25°C时，孵化后大约48 h出现第二龄幼虫，大约24 h后出现三龄幼虫。如果有兴趣在NMJ检查神经变性，二龄幼虫需要受伤。 </li></ol> 3.准备果蝇幼虫机械损伤<ol><li>用镊子或画笔仔细挑选个别幼虫，谨慎不要压缩它。 </li><li>将十只幼虫直接放入含有冷1X PBS或解剖缓冲液的玻璃皿中，以去除任何食物残渣并减缓幼虫运动。 </li></ol> 4. 果蝇幼虫的机械损伤和治疗<ol><li>将每个幼虫置于CO 2麻醉装置上。 </li><li>在解剖显微镜下，仔细将每只幼虫卷入其背侧，以便通过角质层观察节段性神经。 </li><li> 位置大小5镊子从嘴钩开始的长度大约从1/2到2/3。一旦定位，夹住包含5个镊子的节段神经的大约1/3的腹角质层。 <ol><li>为了确保幼虫节段性神经受伤，应用足够的力量粉碎节段性神经，但使角膜完好无损。确定正确的力量，粉碎10只幼虫并确保5小时后的死亡率低于50％。 </li></ol></li><li>损伤后，仔细将幼虫转移到含有约0.5g酵母膏的果汁琼脂平板上。放置每只幼虫，使其前端处于酵母糊上，尽管运动性受到破坏，仍可持续喂食。 注意:为了确保幼虫节段性神经受损，转移到琼脂板后可以观察到破坏的幼虫运动。受伤的幼虫在损伤部位后面有效地瘫痪，但仍然可以移动他们的嘴钩和饲料。受伤后动物的高死亡率是常见的，所以最好比实验所需的动物多20-50％。 </li><li>将含有酵母膏和10只受伤幼虫的琼脂平板放入空的100×15mm培养皿的底部。盖上这个petri板，现在用含有湿纸巾的琼脂板上含有受伤的幼虫，确保纸巾不接触幼虫或琼脂板。放这个培养皿放入更大的气密容器中。 </li><li>在伤后特定时期（6,12或24小时）取回幼虫进行解剖。 注意:为了检查损伤对轴突细胞骨架的即时影响，可以在损伤后直接切除幼虫。为了检查轴突运输缺陷，损伤后约6小时可以解剖幼虫。损伤后约12小时可以观察到泛素化蛋白质的积聚，并且可以观察到损伤后24小时，NMJ处的运动神经元变性。 </li></ol> 5. NMJ神经退行性分析<ol><li>解剖果蝇幼虫NMJ如前所述。 15,16简单地说，销幼虫到的Sylgard板，沿背表面切割，圆角与解剖缓冲一滴销以露出体壁肌肉组织。根据抗体，在4％多聚甲醛（PFA）或Bouin&#39;s Solution中修复幼虫身体用于可视化神经元和肌肉。 注意:如果将荧光蛋白标签（如绿色荧光蛋白（GFP））可视化为Bouin固定剂可能太苛刻，则应使用PFA固定。 </li><li>执行免疫荧光，先前已经描述了图11，13，16，同时标记突触前运动神经元和突触后相邻（SSR）肌肉褶皱。使用荧光共轭辣根过氧化物酶（HRP）（1:300） 17可视化突触前膜和轴突，使用抗Bruchpilot（Brp）抗体（nc82; 1:100; Developmental Studies Hybridoma Bank）染色活性区。用抗Dics Large（Dlg）抗体（1:10,000）同时标记突触后肌肉。 18 </li><li>如前所述，进行定量的NMJ神经变性测定。 8 </suP&gt;，9，10，11，12，13，14简言之，同时可视化对于前和突触后隔室单独NMJs。标记有突触后标记但不突触前标记的任何引物指示神经变性的位点（ 图1 ）。每个NMJ的平均引流次数以及每只动物的NMJ平均数量可以被量化。 </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+Kinase1+controls+synaptic+stability+downstream+of+the+type+II+BMP+receptor.">LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor.</a> Neuron. 47 (5), 695-708 (2005).</li><li>Pielage, J., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+spectrin+is+essential+for+synapse+stabilization.">Presynaptic spectrin is essential for synapse stabilization.</a> Curr Biol. 15 (10), 918-928 (2005).</li><li>Pielage, J., Cheng, L., Fetter, R. D., Carlton, P. M., Sedat, J. W., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+presynaptic+giant+Ankyrin+stabilizes+the+NMJ+through+regulation+or+presynaptic+microtubules+and+transsynaptic+cell+adhesion.">A presynaptic giant Ankyrin stabilizes the NMJ through regulation or presynaptic microtubules and transsynaptic cell adhesion.</a> Neuron. 58 (2), 195-209 (2008).</li><li>Massaro, C. M., Pielage, J., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+that+enhance+synapse+stability+despite+persistent+disruption+of+the+spectrin/ankryin/microtubule+cytoskeleton.">Molecular mechanisms that enhance synapse stability despite persistent disruption of the spectrin/ankryin/microtubule cytoskeleton.</a> J Cell Biol. 187 (1), 101-117 (2009).</li><li>Lincoln, B. L., Alabsi, S. 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作者没有什么可以披露的。

早先描述的神经元机械损伤可以用于诱导果蝇幼虫节段神经的损伤/应激。 4，5，6，14本实验技术先前已经被用于剖析事件导致神经变性的时间序列，以及检查在运动神经元细胞体的转录变化后的损伤。 5，14此外，该技术已被用在微流控芯片结合，以...

神经元变性发生在正常发育过程中，可由自然衰老过程，损伤，压力或疾病状态引起。 果蝇黑腹果蝇是常见的果蝇，提供了一个简单而强大的模型生物体来研究神经变性，因为促进神经元退化的分子机制有显着的相似之处。这些相似之处突出表明，大约70％的疾病相关人类基因具有果蝇同系物。 1另外，研究人类神经变性疾病的许多测定和技术工具已被开发并在果蝇中使用 。 2，3在果蝇中 ，神经肌肉接头（NMJ）允许细胞和电生理学特性的分析，并已被证明是研究神经肌肉病的重要系统由于可见Ñeuron肌肉连接。 2在本研究中，我们描述了果蝇幼虫体内神经元损伤测定，其允许节段性神经的可重复损伤。这种运动神经元损伤导致细胞事件的时间序列，导致NMJ损伤后24小时的神经变性。导致神经变性的可重复损伤运动神经元的能力具有多种应用，例如鉴定退行性过程所需的特定基因，对神经元损伤的转录反应的解剖以及保护性信号级联的分析。 4，5，6这方法也被结合使用微流体来研究神经元变性和再生在活的动物。 7 我们利用建立的定量测定来检查运动神经元的退化离子在果蝇 NMJ机械损伤后。该测定是基于突触前膜和蛋白质的丢失先于以突触后肌肉膜折叠为特征的亚突触网（SSR）的拆卸。 8，9，10，11，12，13该试验允许在突触前神经元已经失去了连接到相邻的突触后肌肉"突触足迹"的量化。退化过程已被证明是在幼虫发育12中进行的，并且不能通过改变的突触发育或发芽来解释。 8，9，10，11，12中的广告使用机械损伤超过预先存在的突变的优点是它允许解剖导致NMJ神经变性的细胞事件的时间序列。 13

<table>
	<tbody>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>CO2 Air Tank</td>
			<td>Tech Air</td>
			<td>UN 1013</td>
			<td>Various tank sizes can be purchased/</td>
		</tr>
		<tr>
			<td>CO2 Anesthetizing Apparatus</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless-steel pins, size 0.1</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SylGard 184 Silicone Elastomer, Base and Curing Agent</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>To pour dissecting plates</td>
		</tr>
		<tr>
			<td>Bouin&#39;s Solution</td>
			<td>Sigma</td>
			<td>HT 10132-1L</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde in PBS</td>
			<td>Affymetrix</td>
			<td>FLY-8030-20</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>Dissecting Stereo MIcroscope</td>
			<td>AmScope</td>
			<td>SM-1BZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>AmScope</td>
			<td>HL150-AY-220V</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- nc82 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82-s</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AF3</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor anti-horseradish peroxidase</td>
			<td>Jackson Immunoresearch</td>
			<td>123-545-021; 123-585-021; 123-605-021</td>
			<td>One can you Alexa Fluor&#174; 488, 594 or 647</td>
		</tr>
		<tr>
			<td>Flystuff Grape Juice Agar Premix</td>
			<td>Genesee Scientific</td>
			<td>47-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Genesee Scientific</td>
			<td>29-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Utility Box</td>
			<td>Fisher Scientific</td>
			<td>03-484C</td>
			<td>Used to create humid chamber for larval recovery</td>
		</tr>
	</tbody>
</table>

a simple neuronal mechanical injury methodology to study drosophila motor neuron degeneration

神经元的退化发生在正常发育过程中，并且在损伤，压力和疾病的反应中发生。神经元变性的细胞标志在人类和无脊椎动物中是非常相似的，驱动这些过程的分子机制也是如此。 果蝇，果蝇 ，提供了一个强大但简单的遗传模型生物体来研究神经变性疾病的细胞复杂性。事实上，约70％的疾病相关人类基因具有果蝇同系物，并且已经使用苍蝇描述了大量的工具和测定来研究人类神经变性疾病。更具体地说， 果蝇中的神经肌肉接头（NMJ）已经被证明是研究神经肌肉疾病的有效系统，因为能够分析神经元和肌肉之间的结构连接。在这里，我们报告在果蝇的 体内运动神经元损伤测定再次诱导NMJ神经退行24h。使用这种方法，我们描述了导致运动神经元变性的细胞事件的时间序列。损伤方法具有多种应用，并且还被用于鉴定神经变性所需的特定基因并解剖对神经元损伤的转录反应。

使用这里提出的程序，我们已经证明机械神经元损伤允许神经变性事件的时间解剖。 14，第 18的事件的序列先前已表征和与细胞骨架的立即破坏，随后轴突贩卖缺陷，泛素化蛋白的积累和随后的神经变性24小时后损伤开始。在损伤前，WT NMJs显示突触前活动区标记Brp与整个NMJ的突触后标记物Dlg相一致（ 图1A ?...

Watch this Scientific Journal Video about A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration at JoVE.com

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

在这里，我们描述了一种简单且广泛可及的方法来伤害果蝇幼虫的节段性神经，可视化和量化三龄幼虫神经肌肉接头（NMJ）运动神经元的神经变性。

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

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Research

JoVE Journal

Neuroscience

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

5.5K 次观看.  Quinnipiac University. 在这里，我们描述了一种简单且广泛可及的方法来伤害果蝇幼虫的节段性神经，可视化和量化三龄幼虫神经肌肉接头（NMJ）运动神经元的神经变性。 

视频: A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

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 ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...