This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

研究果蝇在遵守阿尔伯塔省的研究准则大学。 1.收集飞行<ol><li>收集20苍蝇使用CO 2（克）麻醉并置于含有食品25毫米×95毫米收集瓶中。 </li><li>含蝇水平商店小瓶，以避免在那些在底部积聚小瓶任何液体捕集苍蝇。 </li><li>在22℃在培养箱中孵育苍蝇为至少21小时，在45％的湿度下约15小时。以12小时光照设置孵化器:黑暗周期。 </li></ol> 2.登山分析<ol><li>第二天早上，转移20苍蝇从单一到小瓶250毫升的玻璃量筒。标记滚筒的位置，以保持它恒定日常生活。使用每个基因型一层玻璃圆筒，以防止基因型之间的交叉污染。在每个实验和RO的清洗结束基因型之间优化射孔他们。 <ol><li>进行中的环境光的实验（或红光是否有视力潜在缺陷）在温度和湿度为22℃和40％分别。为了避免昼夜节律惊慌失措，总是在每天的同一时间进行实验。 </li></ol></li><li>密封圆筒的顶部用阻挡膜（蜡膜），以防止任何苍蝇的逃逸（ 图2）。 </li><li>设置在三脚架上的摄像机。在190毫升线的250毫升对焦摄像头量筒（17.5厘米）。 </li><li>计数死蝇数在圆筒的底部，并在食品小瓶中。记录该号码作为死亡率。 </li><li>很轻多次敲击对闭孔泡沫垫气缸以足够的力量以置换苍蝇的内底表面。轻按5 - 10倍，而用另一只手按在相机上记录。 </li><li>按相机上的"录制"按钮。 </li><li>启动VIDEO相机记录和挖掘缸六次在不同的非节奏模式。 </li><li>进行每次试验用于从苍蝇最后抽头的时间2分钟，并记录蝇渡17.5厘米（190毫升）中选择每个时间点（量化每10秒）的高度的数量。注意:毫升标记在缸体会有所不同，从一个气缸模型到另一个视直径。为了避免错误，测量所用每个气缸的高度。 </li><li>一旦审判已经结束，处理苍蝇在95％乙醇。 </li><li>重复步骤2.1至2.9，直到所有的重复每次都过测试，新鲜苍蝇。 注:虽然5次重复可能足以与具有运动有很强的影响的突变，20蝇（200苍蝇）的10次生物学重复，建议检测更小的差异。 </li><li>在实验完成后，洗气瓶在实验室洗碗机和干燥O / N被重新使用。 </li></ol> 3.分析<ol><li> ANALY每个视频泽飞试验。每10秒，记录果蝇通过目标线的总数。 <ol><li>如果一只苍蝇爬上回落或下降，飞为-1和记录算下飞行越过目标线为相同数量的攀升是回落或下降飞。例如，如果在15 日飞落在目标线下方，下一飞越线（16 日飞）被认为是15 日飞，而不是16 个 。 </li></ol></li><li>从苍蝇（20）的总人数减去死亡获得苍蝇停留在试验的数量。在每个时间点，获得蝇目标线以上的分数。 </li><li>绘制每个百分比在每个时间点（参见图3）。 </li><li>在120秒的数据点进行分析的性能和执行学生t检验时2基团存在或ANOVA和一个检测后多重比较（和Bonferroni修改为计划和Tukey计划外协mparisons）。柯尔莫哥洛夫-斯米尔诺夫测试17也进行确定正态性和方差相等，但也给突变组的分布比较控制。 </li><li>呈现的数据过时效，画出蝇的百分比在120秒苍蝇不同年龄的攀爬（2天，1周，2周），以查看是否有一个渐进赤字（ 图4）。 </li></ol>

<ol>
	<li>Auluck, P. K., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+prevention+of+Parkinson+disease+in+Drosophila.">Pharmacological prevention of Parkinson disease in Drosophila.</a> Nature medicine. 8, 1185-1186 (2002).</li><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annual review of neuroscience. 26, 627-656 (2003).</li><li>Bolduc, F. V., Bell, K., Cox, H., Broadie, K. S., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excess+protein+synthesis+in+Drosophila+fragile+X+mutants+impairs+long-term+memory.">Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory.</a> Nature neuroscience. 11, 1143-1145 (2008).</li><li>McBride, S. M., 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=Pharmacological+rescue+of+synaptic+plasticity,+courtship+behavior,+and+mushroom+body+defects+in+a+Drosophila+model+of+fragile+X+syndrome.">Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome.</a> Neuron. 45, 753-764 (2005).</li><li>Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+epilepsy:+bss+is+a+gain-of-function+mutation+in+the+para+sodium+channel+gene+that+leads+to+seizures.">Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures.</a> Genetics. 187, 523-534 (2011).</li><li>Marley, R., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+persistent+Na++current+contributes+to+seizure+in+the+slamdance+bang-sensitive+Drosophila+mutant.">Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant.</a> Journal of neurophysiology. 106, 18-29 (2011).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Mutants+Of+Drosophila+Isolated+By+Countercurrent+Distribution.">Behavioral Mutants Of Drosophila Isolated By Countercurrent Distribution.</a> Proceedings of the National Academy of Sciences of the United States of America. 58, 1112-1119 (1967).</li><li>Ganetzky, B., Flanagan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relationship+between+senescence+and+age-related+changes+in+two+wild-type+strains+of+Drosophila+melanogaster.">On the relationship between senescence and age-related changes in two wild-type strains of Drosophila melanogaster.</a> Experimental gerontology. 13, 189-196 (1978).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Toma, D. P., White, K. P., Hirsch, J., Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+involved+in+Drosophila+melanogaster+geotaxis,+a+complex+behavioral+trait.">Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait.</a> Nature genetics. 31, 349-353 (2002).</li><li>Inagaki, H. K., Kamikouchi, A., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+simple+gravity+sensing+in+Drosophila+melanogaster.">Methods for quantifying simple gravity sensing in Drosophila melanogaster.</a> Nature protocols. 5, 20-25 (2010).</li><li>Gargano, J. W., Martin, I., Bhandari, 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> Experimental gerontology. 40, 386-395 (2005).</li><li>Botella, J. A., 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=The+Drosophila+carbonyl+reductase+sniffer+prevents+oxidative+stress-induced+neurodegeneration.">The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.</a> Current biology : CB. 14, 782-786 (2004).</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>Chakraborty, R., 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=Characterization+of+a+Drosophila+Alzheimer's+disease+model:+pharmacological+rescue+of+cognitive+defects.">Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects.</a> PLoS One. 6, e20799 (2011).</li><li>Orso, G., 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=Disease-related+phenotypes+in+a+Drosophila+model+of+hereditary+spastic+paraplegia+are+ameliorated+by+treatment+with+vinblastine.">Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine.</a> J Clin Invest. 115, 3026-3034 (2005).</li><li>Lehmann, E. L., D'Abrera, H. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nonparametrics : statistical methods based on ranks. , (2006).</li><li>Sherwood, N. T., Sun, Q., Xue, M., Zhang, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+spastin+regulates+synaptic+microtubule+networks+and+is+required+for+normal+motor+function.">Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.</a> PLoS biology. 2, e429 (2004).</li><li>Inlow, J. K., Restifo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+comparative+genetics+of+mental+retardation.">Molecular and comparative genetics of mental retardation.</a> Genetics. 166, 835-881 (2004).</li><li>Palladino, M. J., Hadley, T. J., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+paralytic+mutants+are+enriched+for+those+causing+neurodegeneration+in+Drosophila.">Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in Drosophila.</a> Genetics. 161, 1197-1208 (2002).</li></ol>

The authors have nothing to disclose.

果蝇已经被证明是在帕金森氏病14和其他神经变性病症1,2-极好模型。除了 ​​在果蝇中可用的遗传工具，其基因组中是高度保守的对参与神经障碍19基因。全基因组基因筛查方法（包括全外显子组测序）的出现很可能继续提供与人类运动障碍相关的候选基因较大名单。的治疗这些疾病的发展将需要的动物模型，以增加我们涉及神经变性的早期阶段的病理学的...

破坏性神经变性疾病如帕金森氏病，肌萎缩性侧索硬化，和遗传性痉挛性截瘫日益认识到。不幸的是，大多数这些神经变性疾病的仍然没有治疗方法。的广泛临床应用的全基因组，无偏基因测试，如全基因组测序已导致了越来越多的基因受到牵连在人类运动障碍。尽管这样的进步，从早期到晚期的病理进展，仍然难以在这些疾病。 果蝇提供一个具有遗传工具来研究基因在需求控制的空间和时间的方式。此外， 果蝇已经证明在筛选药物对神经系统疾病，如帕金森氏1，阿尔茨海默2，智力障碍3,4-和癫痫5,6-等等是有用的。我们的目标是建立一个符合成本效益可靠的检测，将允许高通量分析，将仍然非常敏感，可以检测电机性能的微小变化。 有用于量化对果蝇攀登行为的基因突变和/或环境条件的影响几个实验。大多数的测定的利用苍蝇爬，称为负趋地性，或攀登测定的自然趋势。本泽7在1967年提出，用于趋光性的研究逆流装置也可以用来研究gravitaxis。自那时以来，Ganetsky 8和许多其他9 -12已经建立在初始测定。其原理是放置在小瓶中的苍蝇的已知数量和强烈挖掘小瓶在硬表面上，造成苍蝇下降到小瓶的底部。因为它是一个先天行为，苍蝇将试图攀爬到小瓶的顶部，相对的重力。该测定是定量的测量仪表水库有多少苍蝇在一个规定的时间段过去攀上小瓶标记。速度，而不是对苍蝇攀登总数的测量已成为一个可靠的参数和的情况下的苍蝇准则的数目并不显著13中所示的缺陷。 攀登测定在许多神经变性疾病，包括帕金森氏病14的研究证明是有益的。然而，我们注意到，机车的缺陷未必在时间被检测到，其中神经变性是已经看到在病理研究14。因此，使用传统的测定法的可能限制，研究疾病发病的早期阶段的能力。机车缺陷在以后的病理阶段的出现可能反映了其疾病进展是太先进完整的救援。 这就提出了与传统的攀登测定的灵敏度的潜在问题。的俗的电位不能tional爬坡法检测温和机车缺陷可以归因于向其中的苍蝇都需要攀登高度。传统的试验15,16措施蝇的数目，成功地翻越2至5厘米的高度在10至20秒。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

从神经变性疾病造成机车的缺陷可以是疾病的晚期发作症状，随后的几年亚临床变性的，因此目前的治疗策略是不能治愈。通过使用全基因组测序的，越来越多的基因已被确定，以在人类运动的作用。尽管确定这些基因中，它是不知道这些基因如何是至关重要的正常机车运作。因此，可靠的测定法，其利用模式生物以阐明，以便确定治疗性利益的新靶标，这些基因的作用，需要比以往更。我们设计了负趋地性测定在先允许对较温和的缺陷的检测，并具有随着时间的推移，以评估这些缺陷的能力的致敏版本。在玻璃进行测定量筒中，被密封与蜡阻挡膜。通过提高门槛的距离要攀升至17.5Ç米和增加实验的持续时间为2分钟，我们观察到在检测温和流动性功能障碍更大的灵敏度。该测定是成本有效的，不需要大量的训练，以获得高度可重复的结果。这使得它一个很好的技术来筛选候选药物在果蝇突变体运动缺陷。

攀岩是一个强大的和可重复的行为。事实上，有一天老野生型果蝇达到目标的距离爬坡性能迅速（25 - 30秒）。突变果蝇呈现范围从轻微（或延迟）的性能来完成无力攀登到目标。我们说明这个这里有两个不同的突变等位基因。第一个是由一个完全缺失的spastin基因（温泉5.75）18的基因spastin的一个严重的等位基因。在这条线（5.75温泉与TM6b）一天的小苍蝇甚至2分钟后达不到WT爬坡?...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

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

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 ...

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.5K 次观看.  University of Alberta. 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. 

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

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

科研

神经科学

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

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

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

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 ...

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

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

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

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

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

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

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 ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...