我们感谢布莱根妇女医院和哈佛医学院的 Mel Feany 博士对照和表达 果蝇 系的 α-突触核蛋白的善意礼物。我们感谢以下对 Olsen 博士的资助来源：NINDS K08、美国帕金森病协会 George C. Cotzias 奖学金、国防部帕金森病早期研究者奖。

用于该测定的果蝇：对照：nSyb-QF2、nSyb-Gal4/+;α-突触核蛋白果蝇：nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+;关闭后 1 天和 10 天;雄性和雌性（交配和未交配）苍蝇（见 材料表）。
1.准备染色的苍蝇食品
<ol><li>将蓝色软凝胶糊食用色素（见 材料表）与蒸馏水以 1：1 的比例 （v/v） 混合。 注意：使用仅含有不可吸收染料的商业食用色素，因为一些蓝色染料已被证明具有神经保护作用21.</li>
	<li>以 10-15 秒的间隔用微波炉加热由标准玉米面 - 琼脂（见 材料表）组成的苍蝇食品小瓶，直到食物融化成液体或浆液。不要让食物沸腾。</li>
	<li>将染料混合物添加到每瓶食物中，以获得小瓶之间的均匀着色。混合在染料混合物中，直到食物呈均匀的蓝色（图1）。加入一定量的蓝色染料混合物，使食物的颜色饱和，并且小瓶之间的颜色没有变化。 注意： 此步骤必须在微波炉加热后立即完成，因为食物会很快凝固。如果食物凝固，请再次用微波炉加热小瓶。</li>
	<li>将染色食品小瓶风干直至凝固。干燥时将薄纸巾放在小瓶开口上，以防止任何流浪苍蝇落在小瓶中。</li>
	<li>食物冷却凝固后，将用于测试的苍蝇转移到蓝色食物中。 注意：对于窄瓶（25 毫米），建议在每个小瓶中添加 9-14 只苍蝇。对于宽小瓶（28.5 mm），建议在每个小瓶中加入10-20只苍蝇。苍蝇不按性别或交配经验分开，包括雄性和雌性苍蝇的混合种群，包括交配和未交配的雌性。</li>
	<li>将小瓶与苍蝇一起在培养箱中25°C孵育过夜。</li>
</ol>2. 对苍蝇进行成像
注意：此步骤是可选的（图 2）。
<ol><li>第二天早上（时间0），用二氧化碳麻醉代表苍蝇60秒。放置苍蝇，使腹侧朝上。</li>
	<li>使用相机（见 材料表），每小时捕捉苍蝇的图像，以可视化消化系统中剩余的蓝色食物量。 注意：这些苍蝇不应用于测定的定量部分（步骤3），因为麻醉可能会影响结果。</li>
	<li>每小时对新苍蝇进行麻醉和成像。</li>
</ol>3.进行便秘测定
<ol><li>将剩余的（未麻醉的）苍蝇转移到装有标准 果蝇 食物的小瓶中。对每个小瓶进行编号。</li>
	<li>将苍蝇留在25°C的培养箱中60分钟。</li>
	<li>60分钟后，将苍蝇转移到装有标准 果蝇 食物的新小瓶中。从第一组样品瓶开始数据记录。</li>
	<li>在小瓶的长度上画一条虚线，以标记开始计数的点。</li>
	<li>手动计算每个小瓶壁上小圆点的数量。不要计算在食物或小瓶塞上发现的点。</li>
	<li>首先计算每个小瓶壁上的蓝点数量。</li>
	<li>计算每个小瓶壁上不透明的无色点的数量。 注意：每个点是苍蝇粪便，它是尿液和粪便的组合 22.有时，苍蝇可能会走过粪便并留下痕迹，在这种情况下，只计算原始点，而不是痕迹中的每个标记。</li>
	<li>记录每个小瓶的蓝点与点总数的比率。 注意：点总数是小瓶壁上的蓝点数与同一小瓶壁上的无色点数相加。这将用于计算每小时蓝色粪便的百分比。</li>
	<li>重复步骤 3.2-3.8 七次，每小时收集一次数据，总共 8 小时。</li>
</ol>4. 数据分析
<ol><li>绘制每种情况下每小时蓝色粪便的百分比。</li>
	<li>比较条件之间的数据。 注意：统计显著性可以通过 3 种方式确定：通过比较条件之间的各个时间点、测量线随时间变化的斜率或比较曲线下的面积。</li>
</ol>

Constipation Assays in &lt;em&gt;Drosophila&lt;/em&gt; Flies

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作者没有什么可透露的。

该协议中有几个步骤将有助于成功完成测定。首先，重要的是要确保每个小瓶的每轮之间的时间间隔在整个实验过程中保持一致。用数字标记小瓶将有助于避免冗长的基因型描述，从而节省时间。此外，在整个实验过程中，粪便计数方法22 保持一致至关重要。虽然在食物和小瓶塞上可以看到蓝色粪便，但无色粪便则不然。因此，不要计算食物或小瓶塞上的蓝点。
在行为测定中，由于苍蝇行为的波动或影响测定的未知变量，结果总是存在不一致的可能性。我们建议在所有实验中使用相同的 果蝇 培养基、相同的食用色素和相同品牌的小瓶。有趣的是，在几项试验中，观察到苍蝇在下午早些时候排便的频率较低，这可能是由于苍蝇的昼夜节律23.然而，这种行为在对照果蝇和表达α-突触核蛋白的果蝇中是一致的，因此不应引起关注。
如果苍蝇在测定开始时没有排泄蓝色粪便，则可能使用的染料色素沉着不足。在这种情况下，可以相应地增加染料与蒸馏水的比例。也有可能当苍蝇的消化道中只剩下少量蓝色食物时，可能很难确定粪便是淡蓝色还是无色。发生这种情况时，在小瓶后面放一张白纸将有助于确定粪便点的颜色。即使粪便是很浅的蓝色，最好将其记录为蓝色粪便而不是无色粪便。
该检测的一个局限性是它需要手动计数粪便斑点。为了提高高通量筛选的潜力，该方案将来可能会被修改，以允许自动定量多孔板中单个果蝇产生的蓝色粪便斑点。另一个局限性是，尽管 α-突触核蛋白模型有可能发展成为 PD 的前驱模型，但尚未确定存在便秘而没有神经退行性变的最佳时间点。
总之，这种方法提供了一种简单、直接的方法来模拟果蝇模型中的便秘，便秘是一种未被充分研究的非运动性 PD 症状。

帕金森病 （PD） 是一种进行性神经退行性疾病，临床特征是存在运动迟缓、强直和震颤等运动症状，导致显着的发病率1。在病理学上，PD的定义是黑质中多巴胺能神经元的丢失和α-突触核蛋白的错误折叠，导致路易体和路易神经突的形成。帕金森病的发病机制仍然知之甚少，可能是由遗传和环境因素的复杂相互作用引起的 2,3。目前，尚无改善疾病的疗法，部分原因可能是诊断时病理学已处于晚期。研究表明，黑质中超过 60% 的多巴胺能神经元已经因运动症状的发作而丢失，这强调了探索早期疾病检测的潜在生物标志物的必要性4.便秘就是这样一种临床生物标志物，这在帕金森病患者中很常见 5,6，并且可能在运动症状出现之前数年甚至数十年。
尽管 PD 的临床定义基于运动症状，但便秘是几种非运动症状之一，这些症状对症治疗更具挑战性，并导致患者生活质量的显着损害7.代表大脑和肠神经系统之间双向交流的肠脑轴的改变与帕金森病的发病机制有关。在帕金森病患者胃肠道的组织样本中发现了 α-突触核蛋白聚集体8，动物模型表明肠道神经系统中的 α-突触核蛋白聚集体以朊病毒样方式扩散到中枢神经系统9。此外，帕金森病患者在肠道微生物组中表现出异常10，并可能经历过度的肠道炎症11。帕金森病的便秘研究不足，很少有报道的苍蝇 12,13 或啮齿动物14,15 中帕金森氏症相关便秘的模型。
该测定采用果蝇 PD 模型，其中果蝇在泛神经元驱动因子 n-突触反射蛋白的控制下表达人类 α-突触核蛋白基因。这些果蝇表现出 PD 的所有标志性特征，包括 α-突触核蛋白聚集、运动功能障碍和与年龄相关的神经退行性变，导致多巴胺能神经元的丢失16,17。以前的研究引入了测量果蝇粪便输出量以评估肠道功能障碍，量化果蝇粪便并比较各种遗传系的排泄物量，以揭示消化系统的功能差异18,19,20。在这里，我们演示了使用表达人α-突触核蛋白的果蝇的便秘测定。这个简单而有价值的工具可以研究帕金森病的重要非运动症状。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1400 g sucrose</td>
			<td>MP Biomedicals</td>
			<td>904713</td>
			<td></td>
		</tr>
		<tr>
			<td>1800 g dextrose</td>
			<td>MP Biomedicals</td>
			<td>901521</td>
			<td></td>
		</tr>
		<tr>
			<td>2884 g yeast</td>
			<td>MP Biomedicals</td>
			<td>903312</td>
			<td></td>
		</tr>
		<tr>
			<td>428 g agar</td>
			<td>Fisher Scientific</td>
			<td>10253156</td>
			<td></td>
		</tr>
		<tr>
			<td>4600 mL molasses</td>
			<td>Grandma&#39;s Molasses</td>
			<td>7971942</td>
			<td></td>
		</tr>
		<tr>
			<td>68 L water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>680 mL tegosept mix (1200 g tegosept in 6 L ethanol)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6864 g cornmeal</td>
			<td>Pearl Milling</td>
			<td>125045</td>
			<td></td>
		</tr>
		<tr>
			<td>800 mL acid mix (83 mL phosphoric acid in 1 L water + 836 mL propionic acid in 1 L water)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cellSens Standard software</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-Aper</td>
			<td>111ACS200</td>
			<td></td>
		</tr>
		<tr>
			<td>Flugs for wide plastic vials</td>
			<td>Genesee Scientific</td>
			<td>49-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Flystuff wide Drosophila vials, polystyrene</td>
			<td>Genesee Scientific</td>
			<td>32-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td>Version 9.5.1</td>
		</tr>
		<tr>
			<td>Olympus DP23 camera</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus SZX12 Stereo Microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Fisher Scientific</td>
			<td>S25470A</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic Acid</td>
			<td>Fisher Scientific</td>
			<td>A258 - 500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft gel paste food color, Royal blue</td>
			<td>AmeriColor</td>
			<td>202</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Apex</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nSyb-QF2, nSyb-Gal4</td>
			<td>All lines provided by Dr. Mel Feany</td>
			<td>N/A</td>
			<td>Lines are available directly from Dr. Feany&#160;</td>
		</tr>
		<tr>
			<td>nSyb-QF2, nSyb-Gal4, QUAS-alpha synuclein</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>w1118</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring constipation in a drosophila model of parkinson's disease

帕金森病 （PD） 的非运动症状很常见，难以治疗，并且会严重损害生活质量。一种普遍的非运动症状是便秘，它可能比诊断为帕金森病早数年甚至数十年。便秘在帕金森病的动物模型中尚未得到充分探索，并且缺乏特异性治疗。该测定利用了 PD 的 果蝇 模型，其中人 α-突触核蛋白在泛神经元驱动下表达。表达α-突触核蛋白的果蝇具有帕金森病的标志性特征：多巴胺能神经元的丧失、运动障碍和α-突触核蛋白包涵体。
该协议概述了一种研究这些苍蝇便秘的方法。将苍蝇放在带有蓝色添加剂的苍蝇食物上过夜，然后在第二天转移到标准食物中。随后将它们移至装有标准苍蝇食品的新小瓶中，每小时持续 8 小时。在每次转移之前，计算蓝色粪便斑点与小瓶壁上总粪便斑点的百分比。缺乏α-突触核蛋白的对照果蝇在表达α-突触核蛋白的果蝇之前数小时排出所有蓝色染料。此外，蓝色食物从肠道的通过可以通过简单的摄影来监测。该测定的简单性使其能够用于正向遗传或化学筛选，以鉴定 果蝇便秘的修饰剂。

Parkinson&#39;s disease (PD) is a progressive neurodegenerative disorder characterized clinically by the presence of motor symptoms such as bradykinesia, rigidity, and tremor, resulting in significant morbidity1. Pathologically, PD is defined by the loss of dopaminergic neurons in the substantia nigra and the misfolding of alpha-synuclein, leading to the formation of Lewy bodies and Lewy neurites. The pathogenesis of PD remains poorly understood, likely arising from a complex interplay of genetic and environmental factors2,3. Currently, disease-modifying therapies are unavailable, possibly due in part to the advanced stage of pathology present at the time of diagnosis. Studies have shown that more than 60% of dopaminergic neurons in the substantia nigra have already been lost by the onset of motor symptoms, underscoring the need to explore potential biomarkers for early disease detection4. One such clinical biomarker is constipation, which is common in PD patients5,6 and may precede motor symptom onset by years or even decades.
Despite PD&#39;s clinical definition based on motor symptoms, constipation is one of several non-motor symptoms that are more challenging to treat symptomatically and cause significant impairment in patients&#39; quality of life7. Alterations in the gut-brain axis, which represents bidirectional communication between the brain and the enteric nervous system, have been implicated in PD pathogenesis. Alpha-synuclein aggregates have been found in tissue samples from the gastrointestinal tracts of PD patients8, and animal models suggest that alpha-synuclein aggregates in the enteric nervous system spread in a prion-like manner to the central nervous system9. Additionally, PD patients exhibit abnormalities in the gut microbiome10 and may experience excessive gut inflammation11. Constipation in PD has been understudied, with few reported models of Parkinson&#39;s-associated constipation in flies12,13or rodents14,15.
This assay employs a Drosophila model of PD in which flies express the human alpha-synuclein gene under the control of a pan-neuronal driver, n-synaptobrevin. These flies exhibit all the hallmark features of PD, including alpha-synuclein aggregation, motor dysfunction, and age-related neurodegeneration, resulting in the loss of dopaminergic neurons16,17. Previous studies have introduced the measurement of fecal output in flies to assess gut dysfunction, quantifying fly fecal matter and comparing excreta amounts across various genetic lines to reveal functional differences in the digestive system18,19,20. Here, we demonstrate the constipation assay using flies expressing human alpha-synuclein. This simple yet valuable tool enables the study of an important non-motor symptom of PD.

Author Spotlight: Exploring Non-Motor Symptoms in Parkinson&#039;s Disease 

在帕金森病的 果蝇 模型中测量便秘

因为果蝇的腹部是透明的，所以在活的麻醉果蝇中，可以在肠道内看到蓝色食物。可以通过在不同时间点拍摄苍蝇的图像来评估肠道运输的定性差异。在对照果蝇中，蓝色食物会迅速被排出，而在α-突触核蛋白果蝇中，蓝色食物在肠道中停留长达8小时（图2）。
α-突触核蛋白果蝇表现出与年龄相关的神经变性，在闭合后 10 天形成健壮的表型10。在这个时间点，通过比较条件之间的各个时间点，通过测量线随时间变化的斜率或比较曲线下的面积，可以看到与对照果蝇相比肠道运输时间的差异（图3）。在较早的时间点，即闭合后第1天，在对照果蝇和α-突触核蛋白果蝇之间的肠道运输中没有观察到差异，此时运动功能障碍和神经退行性变也尚未存在（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65966/65966fig1v2.jpg"> 图 1：蓝色食品瓶和粪便斑点。 （A） 所有小瓶都含有蓝色食物。此时，苍蝇刚刚被引入蓝色食物，看不到蓝色粪便。（B） 将 w1118 苍蝇转移到新鲜食物中 1 小时后的小瓶，然后取出。左边的插图在（C）中放大，右边的插图在（D）中放大。（C） 由于存在苍蝇食物而被排除在分析之外的区域。（D） 粪便斑点的放大视图，表示蓝色斑点（黑色箭头）和非蓝色斑点（黄色箭头）。 <a href="https://www.jove.com/files/ftp_upload/65966/65966fig1v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65966/65966fig02.jpg"> 图2：染色食品肠道运输随时间变化的可视化。 对照果蝇和在神经元中表达人类α-突触核蛋白的果蝇在染色的食物上过夜，然后在第二天转移到标准培养基中。苍蝇每小时被连续转移到新的标准苍蝇食物中。图中显示了时间零和转印后 2、4、6 和 8 小时的代表性图像。对照组和α-突触核蛋白果蝇在零点时在肠道中显示出类似的蓝色食物斑块。在随后的时间点，对照果蝇的蓝色食物比α-突触核蛋白果蝇少，并且在对照果蝇的8小时时间点，所有蓝色食物都消失了。各蝇系的基因型如下：对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雌性苍蝇用于成像，因为它们的体型较大，更容易可视化腹部。 <a href="https://www.jove.com/files/ftp_upload/65966/65966fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65966/65966fig3v2.jpg"> 图 3：苍蝇闭合后第 10 天的便秘测定。 对照苍蝇和在神经元中表达人类α-突触核蛋白的果蝇在染成蓝色的食物上过夜，然后在第二天转移到标准苍蝇食物中。苍蝇每小时连续转移到新的标准培养基上。（A） 每小时蓝色粪便的百分比。误差线表示标准偏差。N = 每个基因型 6 瓶;每个小瓶含有 9-14 只苍蝇。所有统计分析均在Graphpad Prism中进行（参见 材料表）。使用双因素方差分析计算每个时间点的统计显着性。使用线性回归分析计算线的斜率和斜率之间的差值。（B） 计算每种基因型的曲线下面积。误差线表示标准偏差。各蝇系的基因型如下：对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雄性和雌性苍蝇的数量大致相等。 <a href="https://www.jove.com/files/ftp_upload/65966/65966fig3v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65966/65966fig4v2.jpg"> 图 4：苍蝇闭合后第 1 天的便秘测定。 对照果蝇和在神经元中表达人类α-突触核蛋白的果蝇在染色的食物上过夜，然后在第二天转移到标准培养基中。苍蝇每小时被连续转移到新的标准苍蝇食物中。（A） 每小时蓝色粪便的百分比。误差线表示标准偏差。N = 每种基因型至少 5 瓶;每个小瓶含有 9-14 只苍蝇。所有统计分析均在Graphpad Prism中进行。使用双因素方差分析计算每个时间点的统计显着性。使用线性回归分析计算线的斜率和斜率之间的差值。（B） 计算每种基因型的曲线下面积。误差线表示标准偏差。各蝇系的基因型如下：对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雄性和雌性苍蝇的数量大致相等。 <a href="https://www.jove.com/files/ftp_upload/65966/65966fig4v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Exploring Non-Motor Symptoms in Parkinson's Disease at JoVE.com

该协议提出了一种用于在基于α-突触核蛋白的帕金森病 果蝇 模型中模拟便秘的测定方法。

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom ...

帕金森病的定义是运动迟缓、震颤和僵硬等运动症状。但许多帕金森病患者患有非运动症状，这些症状更难治疗，并导致更大的生活质量损害。这些尚未在该疾病的动物模型中得到很好的研究，这限制了我们为它们寻找新疗法的能力。该协议简单且价格低廉，但可产生可重复的结果。它易于学习，不需要专门的设备。通过这种简单的检测，我们可以评估患有神经退行性变的果蝇的便秘。未来，我们希望开发一种模型，其中苍蝇在神经退行性变发作之前会出现便秘。这将使我们能够更好地了解帕金森病的发病机制。

measuring-constipation-in-a-drosophila-model-of-parkinson-s-disease

Research

Education

JoVE Journal

JoVE Core

Pharmacology

Neuroscience

Measuring Constipation in a Drosophila Model of Parkinson's Disease

Unit 1

Drugs Affecting Gastrointestinal Motility

SCIENTISTS IN ACTION

966 次观看.  University of Pittsburgh. 帕金森病的定义是运动迟缓、震颤和僵硬等运动症状。但许多帕金森病患者患有非运动症状，这些症状更难治疗，并导致更大的生活质量损害。这些尚未在该疾病的动物模型中得到很好的研究，这限制了我们为它们寻找新疗法的能力。该协议简单且价格低廉，但可产生可重复的结果。它易于学习，不需要专门的设备。通过这种简单的检测，我们可以评估患有神经退行性?...

视频: Author Spotlight: Exploring Non-Motor Symptoms in Parkinson's Disease 

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom is constipation, which can precede the diagnosis of PD by years or even decades. Constipation has been underexplored in animal models of PD and lacks specific therapies. This assay utilizes a Drosophila model of PD in which human alpha-synuclein is expressed under a pan-neuronal driver. Flies expressing alpha-synuclein develop the hallmark features of PD: the loss of dopaminergic neurons, motor impairment, and alpha-synuclein inclusions.
This protocol outlines a method for studying constipation in these flies. Flies are placed on fly food with a blue color additive overnight and then transferred to standard food the following day. They are subsequently moved to new vials with standard fly food every hour for 8 h. Before each transfer, the percentage of blue-colored fecal spots compared to the total fecal spots on the vial wall is calculated. Control flies that lack alpha-synuclein expel all the blue dye hours before flies expressing alpha-synuclein. Additionally, the passage of blue-colored food from the gut can be monitored with simple photography. The simplicity of this assay enables its use in forward genetic or chemical screens to identify modifiers of constipation in Drosophila.

This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease.

科研

神经科学

Preparation of Dyed Fly Food and Constipation Assay Assessment in Drosophila

Preparation of Dyed Fly Food and  Constipation Assay Assessment in Drosophila  

To begin, mix the blue soft gel paste food coloring with distilled water. Microwave the food vials containing standard cornmeal agar food until the food has melted into a liquid. Add the dye mixture to each vial of food and mix well until the food is homogeneously blue.Add an amount of blue dye mixture to saturate the color of the food, ensuring no variation in color between the vials. Place a thin paper towel over vial openings during drying to prevent any stray flies from landing in the vial. Once the food is cooled and solidified, transfer the flies to the blue food containing vial.Incubate the vials with flies overnight at 25 degrees Celsius. The next day, transfer the flies to vials containing standard Drosophila food and number each vial. Incubate the flies at 25 degrees Celsius for 60 minutes.After incubation, transfer the flies to new vials with standard Drosophila food. Draw a dotted line down the length of the vial to mark the starting point for counting. Manually count the small, round, opaque and colorless dots on the wall of each vial.Record the ratio of blue dots to the total number of dots for each vial. In control flies, the blue food is expelled relatively quickly while in alpha-synuclein flies, the blue food remains in the gut for up to eight hours. At day 10 post-eclosion, when the alpha-synuclein flies display a robust neurodegenerative phenotype, significant differences in gut transit time compared to control flies were observed.However, no differences in gut transit were observed between control and alpha-synuclein flies on day one post-eclosion.