Name: assessing neurodegenerative phenotypes in drosophila dopaminergic neurons by climbing assays and whole brain immunostaining
Uploaded: 2013-04-24T13:00:00
Description: Watch this Scientific Journal Video about Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining at JoVE.com

飞股票我们利奥Pallanck感谢，恭索莫斯技术援助和P. Sykiotis耶拉西莫斯有益的讨论。这项工作是由美国国立卫生研究院资助的培训补助T32CA009363 MCB

1。惊吓引起的负趋地性含量<ol><li>选择所需的基因型的苍蝇，他们按性别分开，随机分组同伙20-30动物和它们翻转到新的食品瓶，每2-3天;测试每一组的苍蝇（ 即年龄相匹配的队列，队列/基因型）至少每星期一次。 </li><li>负趋地性测试前30分钟，麻醉苍蝇与CO 2，并将其放置在预先标记的透明塑料管有两个空的食品瓶（见材料表 ）加入在他们的开口处用透明胶带（室?...

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	<li>Bayersdorfer, F., Voigt, A., Schneuwly, J., Botella, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine-dependent+neurodegeneration+in+Drosophila+models+of+familial+and+sporadic+Parkinson's+disease.">Dopamine-dependent neurodegeneration in Drosophila models of familial and sporadic Parkinson's disease.</a> Neurobiol. Dis. 40, 113-119 (2010).</li><li>Feany, M. B., Bender, W. 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Drug Targets. 9, 504-523 (2010).</li><li>Lundell, M., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spacial+development+of+serotonin+and+dopamine+neurons+in+the+Drosophila+CNS.">Temporal and spacial development of serotonin and dopamine neurons in the Drosophila CNS.</a> Dev. Biol. 165, 385-396 (1994).</li><li>Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+different+types+of+dopaminergic+neurons+innervate+the+Drosophila+mushroom+body+neuropil:+anatomical+and+physiological+heterogeneity.">Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.</a> Front Neural Circuits. 3, 5 (2009).</li><li>Mizuno, H., Fujikake, N., Wada, K., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Synuclein+Transgenic+Drosophila+As+a+Model+of+Parkinson's+Disease+and+Related+Synucleinopathies.">Alpha-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies.</a> Parkinsons Dis. 2011, 212706 (2011).</li><li>Neckameyer, W. S., Woodrome, S., Holt, B., Mayer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+and+senescence+in+Drosophila+melanogaster.">Dopamine and senescence in Drosophila melanogaster.</a> Neurobiol. Aging. 21, 145-152 (2000).</li><li>Trinh, K., Moore, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+phase+II+detoxification+pathway+suppresses+neuron+loss+in+Drosophila+models+of+Parkinson's+disease.">Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson's disease.</a> J. Neurosci. 28, 465-472 (2008).</li><li>White, K. E., Humphrey, D. M., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopaminergic+system+in+the+aging+brain+of+Drosophila.">The dopaminergic system in the aging brain of Drosophila.</a> Front Neurosci. 4, 205 (2010).</li><li>Whitworth, A. J., Wes, P. D., Pallanck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+pioneer+a+new+approach+to+drug+discovery+for+Parkinson's+disease.">Drosophila models pioneer a new approach to drug discovery for Parkinson's disease.</a> Drug Discov. Today. 11, 119-126 (2006).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat. Protoc. 1, 2110-2115 (2006).</li><li>Barone, M. C., Sykiotis, G. P., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+activation+of+Nrf2+signaling+is+sufficient+to+ameliorate+neurodegenerative+phenotypes+in+a+Drosophila+model+of+Parkinson's+disease.">Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.</a> Dis. Model Mech. 4 (5), 701-707 (2011).</li></ol>

这里描述的检测提供了一个有用的方法来研究特定基因的作用，信号转导通路或小分子化合物在维护DA能神经元的老化，以及在不同的疾病与遗传背景（5审阅）。 惊跳果蝇行为引起的负趋地性，已被广泛用于作为PD-连锁突变的存在下，特别是在不同的遗传背景中的多巴胺神经元的功能的官能读出。在不同的研究中已观察到一些差异（见12，用于更详细?...

这里描述的检测的特异性依赖于使用的Gal4的利用，以达到特定的多巴胺能神经元中的表达3的酪氨酸羟化酶基因的调节序列的飞线。酪氨酸羟化酶催化的第一和多巴胺合成的限速步骤。 TH免疫反应性在成体果蝇脑中多巴胺，TH一个很好的候选，以确定在体内的多巴胺神经元（见实施例6）重叠。此外，比其他Gal4的线（如DdcGal4），其中包含从多巴脱羧酶基因的调节序列，并驱动转基因的表达不仅在DA能神经元更具体的表达模式THGal4，而且在5 -羟色胺能神经元和神经胶质细胞3的子集。 Feany和德尔（2000）首次观察到泛神​​经元的PD联的人α-突触核蛋白基因的表达逐渐丧失站加速的诱导rtle的负趋地在果蝇的行为， 我们已经观察到了类似的结果，使用的DA神经元特异性THGal4线驱动expressionof的α-突触核蛋白基因10，使用这个功能读出Nrf2与途径研究的神经保护作用，在此果蝇的PD模型14。使用此处所描述的具体的测定适于从2和3。 果蝇的大脑包含超过100个DA能神经元，安排至少 ​​在5个不同的集群（PPL1 PPL2，PAL，/ 2 PPM1，PPM3）全脑坐骑可以可视化激光共聚焦显微镜（例如11和7）。然而，不论是否在果蝇脑年龄下降，9号，11多巴胺神经元的数目;年龄依赖性的神经退行性疾病中观察到的苍蝇PD-连锁基因已突变/过度人类疾病模型，目前仍有争议5。 DA能神经元中的表达人类α-共核蛋白就有这样的效果，因此，已被用来作为帕金森病的模型。在这里，我们描述了一个检测DA能神经元计数安装使用全脑TH细胞标记。此法是改编自13和10。

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rabbit anti-tyrosine hydroxylase antibody</td>
			<td>Millipore</td>
			<td>AB152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey TRITC-labeled anti rabbit IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-026-152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Sigma</td>
			<td>D2522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Polystyrene vials</td>
			<td>VWR</td>
			<td>89092-734</td>
			<td>28.5 x 95 mm</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Canon</td>
			<td>PowerShot A3100IS (model)</td>
			<td>12.1 Megapixels 4X Optical Zoom</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 1.5</td>
			<td>VWR</td>
			<td>48366 205 48393 252</td>
			<td>Sizes: 18 x 18 mm, 24 x 60 mm</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 2</td>
			<td>VWR</td>
			<td>48368-040</td>
			<td>Size: 18 x 18 mm</td>
		</tr>
		<tr>
			<td>Dissection dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-01</td>
			<td>Size: 30 mm O.D.</td>
		</tr>
		<tr>
			<td>Dumostar No. 5 tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>72705-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000 (model)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP2 or SP5 (model)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Materials Table.

assessing neurodegenerative phenotypes in drosophila dopaminergic neurons by climbing assays and whole brain immunostaining

果蝇是一种有价值的模式生物，研究在神经系统的衰老和退行性病理过程。飞作为一个实验系统的优势包括其遗传的可追踪性，寿命短，观察和定量分析复杂的行为的可能性。在特定神经元群体的果蝇脑疾病相关的基因的表达，可以用来模拟人类神经变性疾病如帕金森氏症和阿尔茨海默氏症的5。 多巴胺（DA）神经元是最脆弱的神经元群体在人类大脑的老化。在帕金森氏病（PD），最常见的神经退行性运动障碍，加速多巴胺神经元丢失导致一个渐进的和不可逆的运动功能下降。除了年龄和暴露于环境毒素，加剧了特定的基因突变在编码多巴胺神经元丢失或几个基因的启动子区域的。这样的PD相关的等位基因的鉴定果蝇作为模型来研究神经退行性疾病的DA神经元在体内的利用提供了实验依据。例如，表达的PD联的人α-突触核蛋白基因在果蝇 DA能神经元的概括人类疾病的一些特点， 例如逐步多巴胺神经元丢失和运动功能下降，2。因此，这种模式已经被成功地用于识别潜在的治疗靶点，PD 8。 在这里，我们描述了两个实验，通常被用来研究果蝇 DA能神经元年龄相关的神经退行性疾病：攀岩惊吓引起的负趋地响应和整个成人脑酪氨酸羟化酶免疫的基础上安装检测监控DA能神经元的数量在不同的年龄段。在这两种情况下， 在体内表达的UAS吨ransgenes，特别是在DA能神经元，可以实现使用酪氨酸羟化酶（TH）启动子-Gal4的驱动线3，10。

我们这里描述的实验研究的应力的作用，保护Nrf2的途径在果蝇模型的PD 14。此模型依赖于使用一个TH Gal4的驱动器2，10中的多巴胺神经元的人类α-共核蛋白基因的表达。 图1示出了代表性的结果惊吓引起的负趋地性（攀登）检测对TH Gal4的驱动程序的控制下，表达不同的外源基因在雄蝇。测试所有的基因型表现出运动活性下降，随着时间的推?...

Watch this Scientific Journal Video about Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining at JoVE.com

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

在这里，我们描述了两个实验，已建立了学习年龄依赖性的多巴胺（DA）神经元的神经退行性疾病<em&gt;果蝇</em&gt;：攀登/的惊跳诱导负趋地性试验，允许DA能神经元变性，这是用来识别和计数DA能神经元全脑坐骑酪氨酸羟化酶免疫功能的影响研究。

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 ...

Research

JoVE Journal

Neuroscience

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

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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神经科学

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Primary Culture of Mouse Dopaminergic Neurons

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

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Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

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

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

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