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 &lt;em&gt;Drosophila&lt;/em&gt; Flies

<ol>
	<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut-brain+axis:+is+intestinal+inflammation+a+silent+driver+of+Parkinson's+disease+pathogenesis.">The gut-brain axis: is intestinal inflammation a silent driver of Parkinson's disease pathogenesis.</a> NPJ Parkinsons Dis. 3, 3 (2017).</li><li>Pfeiffer, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-motor+symptoms+in+Parkinson's+disease.">Non-motor symptoms in Parkinson's disease.</a> Parkinsonism Relat Disord. 22 (Suppl 1), (2016).</li><li>Klann, E. 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=The+Gut-brain+axis+and+its+relation+to+parkinson's+disease:+A+review.">The Gut-brain axis and its relation to parkinson's disease: A review.</a> Front Aging Neurosci. 13, 782082 (2021).</li><li>Mukherjee, A., Biswas, A., Das, S. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microenvironmental+changes+as+a+potential+trigger+in+Parkinson's+disease+through+the+gut-brain+axis.">Gut microenvironmental changes as a potential trigger in Parkinson's disease through the gut-brain axis.</a> J Biomed Sci. 29 (1), 54 (2022).</li><li>Hawrysh, P. J., 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=PRKN/parkin-mediated+mitophagy+is+induced+by+the+probiotics+Saccharomyces+boulardii+and+Lactococcus+lactis.">PRKN/parkin-mediated mitophagy is induced by the probiotics Saccharomyces boulardii and Lactococcus lactis.</a> Autophagy. 19 (7), 2094-2110 (2023).</li><li>Liu, W., Lim, K. L., Tan, E. 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 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

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.

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

Measuring Constipation in a Drosophila Model of Parkinson's Disease

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

This protocol presents an assay for modeling constipation in an alpha-synuclein based 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 ...

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892 ビュー.  University of Pittsburgh. パーキンソン病は、運動緩慢、振戦、硬直などの運動症状によって定義されます。しかし、パーキンソン病の患者の多くは、治療が難しく、生活の質に大きな障害を引き起こす非運動症状に苦しんでいます。これらは、この病気の動物モデルでは十分に研究されていないため、新しい治療法を見つける能力が制限されています。このプロトコルはシンプルで安価で?...

ビデオ: 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.

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biomarker identification for gender specificity of alzheimer's disease based on the glial transcriptome profiles

Author Spotlight: Exploring Sex-Specific Glial Signatures and Therapeutic Leads for Alzheimer&amp;#39;s Disease

This study analyzed single-nuclei transcriptomes of thirty-three individuals with Alzheimer's disease (AD), revealing sex-specific DEGs in glial cells. Functional enrichment analysis highlighted synaptic, neural, and hormone-related pathways. Key genes, namely NLGN4Y and its regulators, were identified, and potential therapeutic candidates for gender-specific AD were...

Sex-Specific Glial Biomarkers in Alzheimer&amp;#39;s Disease: A Transcriptome Analysis

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auricular acupressure as an adjuvant treatment for wheezing in stable chronic obstructive pulmonary disease 

Author Spotlight: Efficacy of Auricular Pressure Bean Therapy in Reducing Wheezing Symptoms

This protocol describes an effective therapy using auricular acupressure to relieve wheezing symptoms in stable chronic obstructive pulmonary...

Traditional Chinese Medicine Ear Point Therapy for Wheezing in COPD Patients

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acupoint catgut embedding therapy for abdominal obesity treatment

Author Spotlight: Exploring Thread Embedding in Traditional Chinese Medicine for Metabolic Disease Treatment

This study explores acupoint catgut embedding therapy for obesity, covering patient management, material preparation, acupoint selection, surgery, and post-operative...

Acupoint Embedding Therapy for Managing Abdominal Obesity

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dynamic digital biomarkers of motor and cognitive function in parkinson's disease

Dynamic Digital Biomarkers of Motor and Cognitive Function in Parkinson&#039;s Disease

This protocol offers a digitization of portions of traditional clinical tasks commonly used to measure cognition and motor control in Parkinson&#8217;s disease. Clinical tasks are digitized while biophysical rhythms are co-registered from different functional levels of the nervous systems, ranging from voluntary, spontaneous, automatic to...

Dynamic Digital Biomarkers of Motor and Cognitive Function in Parkinson's Disease

signal acquisition, score interpretation, and economics of a non-invasive point-of-care test for coronary artery disease

Author Spotlight: Enhancing Coronary Artery Disease Diagnosis with Non-Invasive Point-of-Care Testing

The methodology to acquire the physiological signal for a Coronary Artery Disease (CAD) test is presented. A method is proposed to interpret the CAD score concerning test positivity and negativity, including the granularities of each. The economics of the test are discussed in the context of the current standard of...

Non-Invasive Point-of-Care Test for Coronary Artery Disease Assessment 

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ginger moxibustion, a non-pharmacological treatment, for diarrhea in patients with chronic obstructive pulmonary disease

Author Spotlight: Exploring Non-Pharmacological Therapies for Chronic Respiratory Diseases &#8212; Linking Intestinal Microbiome Insights to COPD Treatment

Here, we present the method of using ginger moxibustion to treat diarrhea in patients with chronic obstructive pulmonary...

Assessing the Efficacy of Ginger Moxibustion in Diarrhea Management in COPD

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a porcine model of acute respiratory failure with a continuous infusion of oleic acid

Author Spotlight: Exploring Venous Waveforms for Non&amp;#45;Invasive Respiratory Monitoring in Pigs

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory...

Oleic Acid&amp;#45;Induced Acute Respiratory Decompensation in Pigs

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bringing the clinic home: an at-home multi-modal data collection ecosystem to support adaptive deep brain stimulation

Author Spotlight: Automated Deep Brain Stimulation for Parkinson&#039;s Disease - Exploring the Possibilities and Challenges of Home Monitoring 

The protocol shows a prototype of the at-home multi-modal data collection platform that supports research optimizing adaptive deep brain stimulation (aDBS) for people with neurological movement disorders. We also present key findings from deploying the platform for over a year to the home of an individual with Parkinson's...

Optimizing Deep Brain Stimulation for Patients with Neurological Disorders

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abdominal massage to improve motor dysfunction in rats with cerebral palsy

Author Spotlight: Exploring the Potential of Massage Therapy in Cerebral Palsy Using Animal Models 

Here, we present a set of massage manipulations in the rat model of cerebral palsy that can considerably improve the motor function of cerebral palsy...

Chinese Massage Techniques: Enhancing Motor Function in Cerebral Palsy Rats

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the ex vivo preparation of spinal cord slice for the whole-cell patch-clamp recording in motor neurons during spinal cord stimulation

Author Spotlight: Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability...

Understanding Motor Neuron Responses to Spinal Cord Stimulation

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motor imagery performance through embodied digital twins in a virtual reality-enabled brain-computer interface environment

Author Spotlight: Enhancing Neurorehabilitation Through EEG, Motor Imagery, and Virtual Reality

Motor imagery in a virtual reality environment has wide applications in brain-computer interface systems. This manuscript outlines the use of personalized digital avatars that resemble the participants performing movements imagined by the participant in a virtual reality environment to enhance immersion and a sense of body...

Motor Imagery Using VR Digital Avatars in a Brain-Computer Interface System

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non-invasive modulation and robotic mapping of motor cortex in the developing brain

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in...

characterizing the relationship between eye movement parameters and cognitive functions in non-demented parkinson's disease patients with eye tracking

Characterizing the Relationship Between Eye Movement Parameters and Cognitive Functions in Non-demented Parkinson&#039;s Disease Patients with Eye Tracking

Here, we present a protocol to study the relationship between the eye movement parameters and cognitive functions in non-demented Parkinson&#39;s disease patients. The experiment used an eye tracker to measure the saccadic amplitude and fixation duration in a visual search task. The correlation with performance in multi-domain cognitive tasks was subsequently...

Characterizing the Relationship Between Eye Movement Parameters and Cognitive Functions in Non-demented Parkinson's Disease Patients with Eye Tracking

motor imagery brain-computer interface in rehabilitation of upper limb motor dysfunction after stroke

Author Spotlight: Using Motor Imagery Brain-Computer Interface to Improve Motor and Cognitive Function in Stroke Patients

The purpose of this study is to provide an important reference for the standard clinical operation of motor imagery brain-computer interface (MI-BCI) for upper limb motor dysfunction after...

Restoring Post-Stroke Motor Function by Motor Imagery Brain-Computer Interface

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non-nutritive suck parameters measurements using a custom pressure transducer system

Author Spotlight: Implications of Non-Nutritive Sucking on Speech Emergence and Infant Development

The non-nutritive suck (NNS) device can easily collect and quantify NNS features using a pacifier connected to a pressure transducer and recorded through a data acquisition system and laptop. Quantification of NNS parameters can provide valuable insight into a child's current and future...

A Custom-Made Pressure Transducer System to Record Non-Nutritive Suck Bursts

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surgical lumbar sympathectomy in mice

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue...

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

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isolation of mouse pancreatic endothelial cells

Author Spotlight: Exploring the Role of the Pancreatic Vasculature in Pancreas-Biology and Disease Development

This protocol describes the isolation of mouse endothelial cells from whole...

Streamlining Mouse Pancreatic Endothelial Cell Extraction for Disease Studies

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technique for intranasal administration of &#945;-synuclein aggregates

Author Spotlight: Elucidating the Origin of &amp;#945;-Synuclein Propagation from the Olfactory Bulbs in Parkinson&amp;#39;s Disease

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's...

&amp;#945;-Synuclein Propagation: Intranasal Administration in Parkinson&amp;#39;s Disease

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a murine model of hyperlipidemia&#45;induced heart failure with preserved ejection fraction

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&amp;#45;Induced Mouse Model

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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.