We acknowledge Dr. Mel Feany at Brigham and Women&#39;s Hospital and Harvard Medical School for the kind gift of the control and alpha-synuclein expressing Drosophila lines. We acknowledge the following sources of grant support to Dr. Olsen: NINDS K08, American Parkinson Disease Association George C. Cotzias Fellowship, Department of Defense Parkinson&#39;s Disease Early Investigator Award.

Flies used in this assay: control: nSyb-QF2, nSyb-Gal4/+; alpha-synuclein flies: nSyb-QF2, nSyb-Gal4, QUAS-alpha-synuclein/+; 1- and 10-days post eclosion; male and female (mated and unmated) flies (see Table of Materials).
1. Preparing the dyed fly food
<ol>
	<li>Mix blue soft gel paste food coloring (see Table of Materials) with distilled water in a 1:1 ratio (v/v). 
	NOTE: Use commercial food coloring that contains only non-absorbable dyes, as some blue dyes have been shown to have neuroprotective effects21.</li>
	<li>Microwave the vials of fly food consisting of standard cornmeal-agar (see Table of Materials) in 10-15 s intervals until the food has melted into a liquid or slurry. Do not allow food to boil over.</li>
	<li>Add the dye mixture to each vial of food to obtain a uniform coloring between vials. Mix in the dye mixture until the food is homogenously blue (Figure 1). Add an amount of the blue dye mixture such that the color of the food is saturated, and there is no variation in color between the vials. 
	NOTE: This step must be done soon after microwaving, as the food solidifies quickly. If the food solidifies, microwave the vial again.</li>
	<li>Leave the vials of dyed food to air dry until it solidifies. Place a thin paper towel over vial openings while drying to prevent any stray flies from landing in the vial.</li>
	<li>Once the food has cooled and solidified, transfer the flies that will be used for testing to the blue food. 
	&#8203;NOTE: For narrow vials (25 mm), it is recommended that 9-14 flies are added to each vial. For wide vials (28.5 mm), it is recommended that 10-20 flies are added to each vial. Flies are not separated by sex or mating experience and include a mixed population of male and female flies, with both mated and unmated females.</li>
	<li>Incubate the vials with flies overnight at 25 &#176;C in an incubator.</li>
</ol>
2. Imaging the flies
NOTE: This step is optional (Figure 2).
<ol>
	<li>The following morning (time 0), anesthetize the representative flies with carbon dioxide for 60 s. Place the fly so that the ventral side is facing upwards.</li>
	<li>Using a camera (see Table of Materials), capture images of the flies at each hour to visualize the amount of blue food left in the digestive system. 
	&#8203;NOTE: These flies should not be used for the quantitative part of the assay (step 3), as anesthesia may affect the results.</li>
	<li>At every hour, anesthetize and image new flies.</li>
</ol>
3. Performing the constipation assay
<ol>
	<li>Transfer the remaining (non-anesthetized) flies to vials with standard Drosophila food. Number each vial.</li>
	<li>Leave the flies in the incubator at 25 &#176;C for 60 min.</li>
	<li>After 60 min, transfer the flies to new vials with standard Drosophila food. Begin data recording from the first set of vials.</li>
	<li>Draw a dotted line down the length of the vial to mark the point from which counting will start.</li>
	<li>Manually count the number of small, round dots on the wall of each vial. Do not count dots found on the food or vial plug.</li>
	<li>Begin by counting the number of blue dots on the wall of each vial.</li>
	<li>Count the number of opaque, colorless dots on the wall of each vial. 
	NOTE: Each dot is fly excrement, which is a combination of urine and fecal matter22. On occasion, a fly may walk over the excrement and leave a trail, in which case, count only the original dot, not each individual mark in the trail.</li>
	<li>Record the ratio of blue dots to the total number of dots for each vial. 
	NOTE: The total number of dots is the number of blue dots on the wall of a vial added to the number of colorless dots on the wall of the same vial. This will be used to calculate the percent of blue fecal matter per hour.</li>
	<li>Repeat steps 3.2-3.8 seven more times, collecting data every hour across a total of 8 h.</li>
</ol>
4. Data analysis
<ol>
	<li>Graph the percent blue fecal matter per hour for each condition.</li>
	<li>Compare the data between conditions. 
	NOTE: Statistical significance can be determined in 3 ways: by comparing individual timepoints between conditions, by measuring the slope of the line over time, or by comparing the area under the curve.</li>
</ol>

Constipation Assays in Drosophila Flies

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	<li>Kalia, L. V., Lang, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease.">Parkinson's disease.</a> Lancet. 386 (9996), 896-912 (2015).</li><li>Bloem, B. R., Okun, M. S., Klein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease.">Parkinson's disease.</a> Lancet. 397 (10291), 2284-2303 (2021).</li><li>Ball, N., Teo, W. P., Chandra, S., Chapman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+and+the+environment.">Parkinson's disease and the environment.</a> Front Neurol. 10, 218 (2019).</li><li>Zhou, J., Li, J., Papaneri, A. B., Kobzar, N. P., Cui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neuron+challenge+test+for+early+detection+of+Parkinson's+disease.">Dopamine neuron challenge test for early detection of Parkinson's disease.</a> NPJ Parkinsons Dis. 7 (1), 116 (2021).</li><li>Ueki, A., Otsuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+style+risks+of+Parkinson's+disease:+association+between+decreased+water+intake+and+constipation.">Life style risks of Parkinson's disease: association between decreased water intake and constipation.</a> J Neurol. 251 (Suppl 7), vII18-vII23 (2004).</li><li>Houser, M. C., Tansey, M. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+dysfunction+in+Parkinson's+disease.">Gut dysfunction in Parkinson's disease.</a> World J Gastroenterol. 22 (25), 5742-5752 (2016).</li><li>Yemula, N., Dietrich, C., Dostal, V., Hornberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+and+the+gut:+symptoms,+nutrition,+and+microbiota.">Parkinson's disease and the gut: symptoms, nutrition, and microbiota.</a> J Parkinsons Dis. 11 (4), 1491-1505 (2021).</li><li>Chen, S. J., Lin, C. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestine-derived+&#945;-synuclein+initiates+and+aggravates+pathogenesis+of+Parkinson's+disease+in+Drosophila.">Intestine-derived &#945;-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila.</a> Transl Neurodegener. 11 (1), 44 (2022).</li><li>Rota, L., 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=Constipation,+deficit+in+colon+contractions+and+alpha-synuclein+inclusions+within+the+colon+precede+motor+abnormalities+and+neurodegeneration+in+the+central+nervous+system+in+a+mouse+model+of+alpha-synucleinopathy.">Constipation, deficit in colon contractions and alpha-synuclein inclusions within the colon precede motor abnormalities and neurodegeneration in the central nervous system in a mouse model of alpha-synucleinopathy.</a> Transl Neurodegener. 8, 5 (2019).</li><li>Diwakarla, S., 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=ATH434+reverses+colorectal+dysfunction+in+the+A53T+mouse+model+of+Parkinson's+disease.">ATH434 reverses colorectal dysfunction in the A53T mouse model of Parkinson's disease.</a> J Parkinsons Dis. 11 (4), 1821-1832 (2021).</li><li>Ordonez, D. G., Lee, M. K., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-synuclein+Induces+mitochondrial+dysfunction+through+spectrin+and+the+actin+cytoskeleton.">&#945;-synuclein Induces mitochondrial dysfunction through spectrin and the actin cytoskeleton.</a> Neuron. 97 (1), 108.e6-124.e6 (2018).</li><li>Olsen, A. L., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+&#945;-synuclein+promotes+neurodegeneration+characterized+by+a+distinct+transcriptional+program+in+vivo.">Glial &#945;-synuclein promotes neurodegeneration characterized by a distinct transcriptional program in vivo.</a> Glia. 67 (10), 1933-1957 (2019).</li><li>Cognigni, P., Bailey, A. P., Miguel-Aliaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neurons+and+systemic+signals+couple+nutritional+and+reproductive+status+with+intestinal+homeostasis.">Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis.</a> Cell Metab. 13 (1), 92-104 (2011).</li><li>Urquhart-Cronish, M., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interplay+in+Drosophila+melanogaster:+chronic+nutritional+deprivation+in+larval+life+affects+adult+fecal+output.">Gene-environment interplay in Drosophila melanogaster: chronic nutritional deprivation in larval life affects adult fecal output.</a> J Insect Physiol. 69, 95-100 (2014).</li><li>Popovic, 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=Blocking+dPerk+in+the+intestine+suppresses+neurodegeneration+in+a+Drosophila+model+of+Parkinson's+disease.">Blocking dPerk in the intestine suppresses neurodegeneration in a Drosophila model of Parkinson's disease.</a> Cell Death Dis. 14 (3), 206 (2023).</li><li>Poteet, E., 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=Neuroprotective+actions+of+methylene+blue+and+its+derivatives.">Neuroprotective actions of methylene blue and its derivatives.</a> PLoS One. 7 (10), e48279 (2012).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+digestive+tract+of+Drosophila+melanogaster.">Anatomy and physiology of the digestive tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Schlichting, 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=A+neural+network+underlying+circadian+entrainment+and+photoperiodic+adjustment+of+sleep+and+activity+in+Drosophila.">A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila.</a> J Neurosci. 36 (35), 9084-9096 (2016).</li></ol>

The authors have nothing to disclose.

There are several steps in this protocol that will aid in the successful completion of the assay. Firstly, it is important to ensure that the time intervals between each round for each vial are consistent throughout the experiment. Labeling the vials with numbers will help to avoid the need for lengthy genotype descriptions, saving time. Additionally, it is crucial that the method for counting fecal matter22 remains consistent throughout the experiment. While blue fecal matter is visible on the food and vial plug, colorless fecal matter is not. Therefore, do not count the blue dots on the food or vial plug.
With behavioral assays, there is always a possibility of inconsistencies in results due to fluctuations in fly behavior or unknown variables affecting the assay. We recommend using the same Drosophila media, the same food dye, and the same brand of vials for all experiments. Interestingly, in several trials, it was observed that flies tend to defecate less frequently in the early afternoon, possibly due to the flies' circadian rhythm23. However, this behavior is consistent in both control flies and flies expressing alpha-synuclein, so it should not raise concerns.
If the flies do not excrete blue fecal matter at the beginning of the assay, it is possible that the dye used is not pigmented enough. In this case, one can increase the ratio of dye to distilled water accordingly. It is also possible that when there is only a small amount of blue food left in the fly's digestive tract, it may be difficult to determine whether the fecal matter is pale blue or colorless. When this occurs, placing a white sheet of paper behind the vial will help determine the color of the fecal dot. Even if the fecal matter is very light blue, it is best to record it as blue fecal matter rather than colorless fecal matter.
One limitation of this assay is that it requires manual counting of fecal spots. To improve the potential for high-throughput screening, this protocol may be modified in the future to allow for automated quantification of blue fecal spots produced by individual flies in multi-well plates. Another limitation is that, although the alpha-synuclein model has the potential to be developed into a prodromal model of PD, an optimal timepoint at which constipation is present without neurodegeneration has not yet been identified.
In summary, this method offers a simple, straightforward approach to modeling constipation, an understudied non-motor PD symptom, in a Drosophila model of PD.

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.

<table><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&#039;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&amp;nbsp;</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>&lt;em&gt;&lt;strong&gt;Drosophila Stocks&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;nSyb-QF2, nSyb-Gal4&lt;/em&gt;</td><td>All lines provided by Dr. Mel Feany</td><td>N/A</td><td>Lines are available directly from Dr. Feany&amp;nbsp;</td></tr><tr><td>&lt;em&gt;nSyb-QF2, nSyb-Gal4, QUAS-alpha synuclein&lt;/em&gt;</td><td></td><td>N/A</td><td></td></tr><tr><td>&lt;em&gt;w1118&lt;/em&gt;</td><td></td><td>N/A</td><td></td></tr></tbody></table>

measuring constipation in a drosophila model of 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.

Because the Drosophila abdomen is transparent, blue food can be visualized inside the gut in living anesthetized flies. Qualitative differences in gut transit can be assessed by taking images of the flies at various time points. In control flies, the blue food is quickly expelled, whereas in alpha-synuclein flies, the blue food remains present in the gut for as long as 8 hours (Figure 2).
The alpha-synuclein flies display age-related neurodegeneration, with a robust phenotype developing by 10 days post-eclosion10. At this timepoint, differences in gut transit time when compared to control flies are seen by comparing individual timepoints between conditions, by measuring the slope of the line over time, or by comparing the area under the curve (Figure 3). No differences are observed in gut transit between control and alpha-synuclein flies at an earlier timepoint, day 1 post-eclosion, when motor dysfunction and neurodegeneration are also not yet present (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65966/65966fig1v2.jpg" /> 
Figure 1: Blue food vials and fecal spots. (A) All vials contain blue-colored food. At this point in time, flies have just been introduced to the blue food, and no blue fecal matter is visible. (B) A vial after w1118 flies have been transferred to fresh food for 1 h, then removed. The left inset is enlarged in (C), and the right inset is enlarged in (D). (C) An area excluded from the analysis due to the presence of fly food. (D) Enlarged view of fecal spots, indicating blue (black arrows) and non-blue spots (yellow arrows). <a href="https://www.jove.com/files/ftp_upload/65966/65966fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65966/65966fig02.jpg" /> 
Figure 2: Visualization of gut transit of the dyed food over time. Control flies and flies expressing human alpha-synuclein in neurons were left on blue-dyed food overnight and then transferred to standard media the next day. Flies were serially transferred to new standard fly food every hour. Representative images at time zero and 2, 4, 6, and 8 h after transfer are shown. Control and alpha-synuclein flies show similar patches of blue food in the gut at time zero. At subsequent timepoints, control flies have less blue food than alpha-synuclein flies, and all blue food is gone at the 8 h timepoint in control flies. The genotypes for each fly line are as follows: control (nSyb-QF2, nSyb-Gal4/+); alpha-synuclein flies (nSyb-QF2, nSyb-Gal4, QUAS-alpha-synuclein/+). Female flies are used for imaging due to their easier ability to visualize the abdomen because of their larger size. <a href="https://www.jove.com/files/ftp_upload/65966/65966fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65966/65966fig3v2.jpg" /> 
Figure 3: Constipation assay in day 10 post-eclosion flies. Control flies and flies expressing human alpha-synuclein in neurons were left on blue-dyed food overnight and then transferred to standard fly food the next day. Flies were serially transferred to new standard media every hour. (A) Percent blue fecal matter per hour. Error bars represent standard deviation. N = 6 vials per genotype; each vial contained 9-14 flies. All statistical analyses were performed in Graphpad Prism (see Table of Materials). Statistical significance at each timepoint was calculated using two-way ANOVA. The slope of the line and the difference between slopes were calculated using the linear regression analysis. (B) The area under the curve for each genotype was calculated. Error bars represent standard deviation. The genotypes for each fly line are as follows: control (nSyb-QF2, nSyb-Gal4/+); alpha-synuclein flies (nSyb-QF2, nSyb-Gal4, QUAS-alpha-synuclein/+). Both male and female flies are included in roughly equal numbers. <a href="https://www.jove.com/files/ftp_upload/65966/65966fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65966/65966fig4v2.jpg" /> 
Figure 4: Constipation assay in day 1 post-eclosion flies. Control flies and flies expressing human alpha-synuclein in neurons were left on blue-dyed food overnight and then transferred to standard media the next day. Flies were serially transferred to new standard fly food every hour. (A) Percent blue fecal matter per hour. Error bars represent standard deviation. N = minimum 5 vials per genotype; each vial contained 9-14 flies. All statistical analyses were performed in Graphpad Prism. Statistical significance at each timepoint was calculated using two-way ANOVA. The slope of the line and the difference between slopes were calculated using the linear regression analysis. (B) The area under the curve for each genotype was calculated. Error bars represent standard deviation. The genotypes for each fly line are as follows: control (nSyb-QF2, nSyb-Gal4/+); alpha-synuclein flies (nSyb-QF2, nSyb-Gal4, QUAS-alpha-synuclein/+). Both male and female flies are included in roughly equal numbers. <a href="https://www.jove.com/files/ftp_upload/65966/65966fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

Measuring Constipation in a Drosophila Model of 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 ...

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

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1.4K Views.  University of Pittsburgh. This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease. 

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

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Genetics

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Behavior

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.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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.

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

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

Measuring Constipation in a Drosophila Model of Parkinson's Disease

Summary

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Chapters in this video

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

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

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

A Simple Technique to Assay Locomotor Activity in Drosophila

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

Mass Histology to Quantify Neurodegeneration in Drosophila

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Measuring Crop Motility and Food Passaging in Drosophila