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（室尺寸：19厘米×2.85厘米）。在我们的分析中，我们通常使用25苍蝇/管。 注意：从麻醉中恢复的时间可从几分钟到几个小时的变化（ 例如，O / N），应优化测试的特定基因型。 </li><li>马克不同的高度（从1到20厘米），在一个垂直的表面活性一片吸墨纸和磁带的文件E，垂直于板凳上。 </li><li>在前面的纸张，将反应管：管应该是所有对齐，并尽可能接近纸张，以便精确的高度测量。 </li><li>管前，将数码相机（见“特种设备”表）的距离为20-30厘米，从纸张上的立场（ 如枪头盒），选择“电影”选项，并开始记录，在这个时候，你也应该启动一个定时器，以保持轨道之间的时间间隔连续试验。 </li><li>让苍蝇为几秒钟（ 例如 10秒）通过敲击管收集在试管底部的。这一步应该一致（ 即相同的时间，相同的强度，同一个运营商）进行整个实验。 </li><li>记录果蝇的运动，持续10秒的数码相机。 </li><li>重复步骤1.5-1.7 14倍，在一分钟的间隔。 </li><li>分析数据： <ol type="a"><li>数一数，上面的苍蝇2厘米10秒大关之后，通过视觉检查的记录电影。 注：在我们的实验条件下，果蝇的负趋地行为没有最后超越了前10秒后，令人吃惊的（ 即苍蝇开始在各个方向移动，10秒后他们惊人的）。控制苍蝇（ 即 1周龄的苍蝇表达THGal4的驱动，看到传说中的图1），在这个时间窗口攀升的最小距离为2厘米。因此，我们使用了2厘米，分析登山活动的苍蝇不同年龄，不同基因型10秒后启动的行为作为参考。这些参数可能需要调整到帐户为行为试验苍蝇的差异（例如，由于不同的遗传背景或实验室条件下）。请注意，在我们的实验条件下，所有的基因型测试表现较差或类似的控制基因型的苍蝇。 </li><li>取平均的结果，从15个试验。 </li><li>测试每种基因型的独立队列的数量快速平均值作为在管内蝇的总数（=爬％活性）的百分比，重复的次数（N）由下式给出</li><li>评估不同基因型之间的统计学意义随着时间的推移。这可以通过与学生的t检验（当比较两个基因型）或2路单因素方差分析，随后由邦费罗尼事后测试（当进行多重比较）使用商业软件，如GraphPad（GraphPad软件，Inc.La拉霍亚， CA，USA）。 </li></ol></li></ol> 注意 进行检测，在同一时间的一天，在室温下，在相同的光照条件下。苍蝇在一个12小时的光/暗周期环境保持最好控制昼夜节律对果蝇的自发行为的效果。 2。酪氨酸羟化酶免疫全脑底座的含量解决方案和缓冲区固定液：4％多聚甲醛（PFA）在磷酸盐缓冲盐水（PBS） 洗涤缓冲液：0.1％的Triton X-100的PBS中阻断缓冲液：0.1 M，pH为7.5的Tris-HCl，0.15M的NaCl，0.1％的Triton X-100和0.5％牛血清白蛋白安装媒体的Mowiol DABCO（见在哈洛E，D.巷抗体实验室手册，冷泉港实验室出版社，冷泉港，NY，USA，10:418（1988协议）） 程序<ol><li>约1分钟，除去角质层蜡填充用70％乙醇在解剖盘认苍蝇。 </li><li>转让过得充满了冰冷的PBS到另一个解剖盘。 </li><li>解剖大脑： <ol type="a"><li>将飞腹侧（可选：删除的翅膀和腿）。 </li><li>拉人头离身：使用一个设置钳持有的身体和另一个到角质层下的眼睛，以防止损坏大脑</li><li>删除长鼻。 </li><li>保持处于打开状态后，取出长鼻的狭缝的相对侧上的头的角质层，并在相反的方向拉，直到大脑中删除从头部日本。 </li><li>从大脑中删除所有的角质层。 </li><li>卸下所有充气的气管组织，这将阻止大脑漂浮在以下孵化步骤。 </li></ol></li><li>关的P-200的移液管尖的前端切几毫米，用0.1％的Triton X-100/PBS溶液和平衡</li><li>使用制备的P-200移液管尖，脑转移到0.5ml的Eppendorf管中填充用0.5ml的4％PFA / PBS中，并保持在冰上，直到完成剥离。 </li><li>修复的大脑在室温下20分钟：把Eppendorf管机架和机架放置一个nutator申请一个温柔的旋转。 </li><li>删除固定液使用P-1000微量移液器，加0.5毫升0.1％的Triton X-100/PBS的，颠倒混匀，我们一起孵育1分钟;重复这种洗涤步骤一次。 </li><li>删除缓冲区第二次洗涤，加0.5毫升0.1％的Triton X-100/PBS的，让在室温下孵育20分钟，重复此步骤两次。 </li><li>卸下清洗缓冲液，并让大脑在封闭缓冲液孵育1小时，在RT（0.1中号的Tris-HCl，pH值7.5，0.15M的NaCl，0.1％的Triton X-100和0.5％BSA）。 </li><li>孵育的大脑与封闭液1:100稀释的抗TH抗体（见“表特定的试剂”），两天，在4°C，轻轻旋转（见第2.6步）。 </li><li>重复洗涤步骤2.7。和2.8 .. </li><li>两天的大脑平衡与二次antibodyin封闭液1:200稀释，在4°C，轻轻旋转。 </li><li>重复洗涤步骤2.7。和2.8 .. </li><li> 如图2A所示，准备安装滑轨： <ol type="a"><li>一西元2×25微升滴安装媒体的盖玻璃（24×60毫米，1.5）。 </li><li>将两个玻璃盖片（尺寸为18×18毫米，第2期）上的安装媒体留在中间的间隙的滑动，让干。 </li></ol> 注意：您在舞台上共聚焦显微镜检查样品架。通过附加一个22×22毫米1.5玻璃盖的端部之一（使用指甲油和磁带），就可以使玻璃盖同样大小的25×75毫米的幻灯片。 </li><li>取出洗涤液，加入50微升的安装介质，并让大脑平衡上下吹打。 </li><li>使用切P-200移液管尖（见步骤2.4），脑转移两个盖玻片之间的空间中滑动并与盖玻片覆盖。 1.5（ 见图2A;定位在幻灯片上看到的大脑7）。 注：样品被安装在两个盖玻片之间，以允许可视化的前，再行rior一侧大脑。 </li><li>从一个角落里，除去所有的空气填充整个空间安装媒体之间的封面眼镜吸管，让干燥和密封用指甲油。 </li><li>为了评估特定集群中的多巴胺能神经元的数目，获得了一系列用1.0μm的步长用激光共聚焦显微镜（解剖识别在果蝇成人脑的多巴胺能集群的7）沿z轴的光学部分。 </li></ol>

<ol>
	<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. 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+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature. 404, 394-398 (2000).</li><li>Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+in+Drosophila+dopaminergic+cells+using+regulatory+sequences+from+tyrosine+hydroxylase.">Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase.</a> J. Neurobiol. 54, 618-627 (2003).</li><li>Gargano, J. W., Martin, I., Bandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>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=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol. Disord. 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>

我们声明，我们没有竞争经济利益。

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 ...

assessing-neurodegenerative-phenotypes-drosophila-dopaminergic

Research

JoVE Journal

Neuroscience

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

19.0K 次观看.  University of Rochester Medical Center. 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果蝇：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。

视频: 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

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

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. ...

科研

神经科学

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

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

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

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

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

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 ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

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

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

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

嗅觉测定的神经退行性疾病的小鼠模型

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

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

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

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

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

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

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

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

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

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

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

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

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

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.