Name: real-time void spot assay
Uploaded: 2023-02-10T12:00:00
Description: Watch this Scientific Journal Video about Real-Time Void Spot Assay at JoVE.com

This work was supported by an NIH grant R01DK119183 (to G.A. and M.D.C.), a pilot project award through P30DK079307 (to M.G.D.), an American Urology Association Career Development award and a Winters Foundation grant (to N.M.), and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).

Urothelial&#160;Piezo1/2 double knockout mice (Pz1/2-KO, genotype: Piezo1fl/fl;Piezo2fl/fl;Upk2CRE+/-) and controls (Pz1/2-C, genotype: Piezo1fl/fl; Piezo2fl/fl; Upk2CRE-/-) were generated in-house from parental strains obtained from the Jax laboratories (Piezo1fl/fl strain # 029213; Piezo2fl/fl strain # 027720; Upk2CRE+/- strain # 029281). Both female (1.5&#8211;3 month...

<ol>
	<li>Dalghi, M. G., Montalbetti, N., Carattino, M. D., Apodaca, 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+urothelium:+life+in+a+liquid+environment.">The urothelium: life in a liquid environment.</a> Physiological Reviews. 100 (4), 1621 (2020).</li><li>de Groat, W. C., Griffiths, D., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+the+lower+urinary+tract.">Neural control of the lower urinary tract.</a> Comprehensive Physiology. 5 (1), 327 (2015).</li><li>Dalghi, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+for+PIEZO1+and+PIEZO2+in+urothelial+mechanotransduction+and+lower+urinary+tract+interoception.">Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.</a> JCI Insight. 6 (19), (2021).</li><li>Montalbetti, N., 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=Bladder+infection+with+uropathogenic+Escherichia+coli+increases+the+excitability+of+afferent+neurons.">Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.</a> American Journal of Physiology. Renal Physiology. 322 (1), 1 (2022).</li><li>Montalbetti, N., 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=Increased+urothelial+paracellular+transport+promotes+cystitis.">Increased urothelial paracellular transport promotes cystitis.</a> American Journal of Physiology. Renal Physiology. 309 (12), 1070 (2015).</li><li>Montalbetti, N., 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=Urothelial+tight+junction+barrier+dysfunction+sensitizes+bladder+afferents.">Urothelial tight junction barrier dysfunction sensitizes bladder afferents.</a> eNeuro. 4 (3), (2017).</li><li>Montalbetti, N., Stocker, S. D., Apodaca, G., Bastacky, S. I., Carattino, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+K++promotes+irritative+voiding+symptoms+and+pain+in+the+face+of+urothelial+barrier+dysfunction.">Urinary K+ promotes irritative voiding symptoms and pain in the face of urothelial barrier dysfunction.</a> Scientific Reports. 9 (1), 5509 (2019).</li><li>Kim, A. K., Hamadani, C., Zeidel, M. L., Hill, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urological+complications+of+obesity+and+diabetes+in+males+and+females+of+three+mouse+models:+temporal+manifestations.">Urological complications of obesity and diabetes in males and females of three mouse models: temporal manifestations.</a> American Journal of Physiology. Renal Physiology. 318 (1), 160 (2020).</li><li>Bartolone, S. N., 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=Micturition+defects+and+altered+bladder+function+in+the+klotho+mutant+mouse+model+of+aging.">Micturition defects and altered bladder function in the klotho mutant mouse model of aging.</a> American Journal of Clinical and Experimental Urology. 8 (3), (2020).</li><li>de Rijk, M. 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=Aging-associated+changes+in+oxidative+stress+negatively+impacts+the+urinary+bladder+urothelium.">Aging-associated changes in oxidative stress negatively impacts the urinary bladder urothelium.</a> International Neurourology Journal. 26 (2), 111 (2022).</li><li>Coyne, K. 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=The+prevalence+of+lower+urinary+tract+symptoms+(LUTS)+and+overactive+bladder+(OAB)+by+racial/ethnic+group+and+age:+results+from+OAB-POLL.">The prevalence of lower urinary tract symptoms (LUTS) and overactive bladder (OAB) by racial/ethnic group and age: results from OAB-POLL.</a> Neurourology and Urodynamics. 32 (3), 230 (2013).</li><li>Wittig, L., Carlson, K. V., Andrews, J. M., Crump, R. T., Baverstock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+bladder+dysfunction:+a+review.">Diabetic bladder dysfunction: a review.</a> Urology. 123, (2019).</li><li>Irwin, D. 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=Population-based+survey+of+urinary+incontinence,+overactive+bladder,+and+other+lower+urinary+tract+symptoms+in+five+countries:+results+of+the+EPIC+study.">Population-based survey of urinary incontinence, overactive bladder, and other lower urinary tract symptoms in five countries: results of the EPIC study.</a> European Urology. 50 (6), 1314 (2006).</li><li>Bogart, L. M., Berry, S. H., Clemens, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symptoms+of+interstitial+cystitis,+painful+bladder+syndrome+and+similar+diseases+in+women:+a+systematic+review.">Symptoms of interstitial cystitis, painful bladder syndrome and similar diseases in women: a systematic review.</a> The Journal of Urology. 177 (2), 450 (2007).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+tract+infection+syndromes:+occurrence,+recurrence,+bacteriology,+risk+factors,+and+disease+burden.">Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden.</a> Infectious Disease Clinics of North America. 28 (1), 1 (2014).</li><li>Fall, M., Logadottir, Y., Peeker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+cystitis+is+bladder+pain+syndrome+with+Hunner's+lesion.">Interstitial cystitis is bladder pain syndrome with Hunner's lesion.</a> International Journal of Urology. 21, 79 (2014).</li><li>Birder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+bladder,+cystitis+and+nerve/urothelial+interactions.">Urinary bladder, cystitis and nerve/urothelial interactions.</a> Autonomic Neuroscience: Basic &amp; Clinical. 182, 89 (2014).</li><li>Rosen, J. M., Klumpp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+pain+from+urinary+tract+infection.">Mechanisms of pain from urinary tract infection.</a> International Journal of Urology. 21 Suppl. 1, 26 (2014).</li><li>Birder, 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=Neural+control+of+the+lower+urinary+tract:+peripheral+and+spinal+mechanisms.">Neural control of the lower urinary tract: peripheral and spinal mechanisms.</a> Neurourology and Urodynamics. 29 (1), 128 (2010).</li><li>Desjardins, C., Maruniak, J. A., Bronson, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+rank+in+house+mice:+differentiation+revealed+by+ultraviolet+visualization+of+urinary+marking+patterns.">Social rank in house mice: differentiation revealed by ultraviolet visualization of urinary marking patterns.</a> Science. 182 (4115), 939 (1973).</li><li>Sugino, Y., 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=Voided+stain+on+paper+method+for+analysis+of+mouse+urination.">Voided stain on paper method for analysis of mouse urination.</a> Neurourology and Urodynamics. 27 (6), 548 (2008).</li><li>Hill, W. G., Zeidel, M. L., Bjorling, D. E., Vezina, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Void+spot+assay:+recommendations+on+the+use+of+a+simple+micturition+assay+for+mice.">Void spot assay: recommendations on the use of a simple micturition assay for mice.</a> American Journal of Physiology. Renal Physiology. 315 (5), (2018).</li><li>Rajandram, 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=Intact+urothelial+barrier+function+in+a+mouse+model+of+ketamine-induced+voiding+dysfunction.">Intact urothelial barrier function in a mouse model of ketamine-induced voiding dysfunction.</a> American Journal of Physiology. Renal Physiology. 310 (9), (2016).</li><li>Ruetten, H., 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+uropathogenic+E.+coli+UTI89+model+of+prostatic+inflammation+and+collagen+accumulation+for+use+in+studying+aberrant+collagen+production+in+the+prostate.">A uropathogenic E. coli UTI89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate.</a> American Journal of Physiology. Renal Physiology. 320 (1), 31 (2021).</li><li>Wegner, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Void+spot+assay+procedural+optimization+and+software+for+rapid+and+objective+quantification+of+rodent+voiding+function,+including+overlapping+urine+spots.">Void spot assay procedural optimization and software for rapid and objective quantification of rodent voiding function, including overlapping urine spots.</a> American Journal of Physiology. Renal Physiology. 315 (4), (2018).</li><li>Wood, R., Eichel, L., Messing, E. M., Schwarz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+noninvasive+measurement+of+cyclophosphamide-induced+changes+in+murine+voiding+frequency+and+volume.">Automated noninvasive measurement of cyclophosphamide-induced changes in murine voiding frequency and volume.</a> The Journal of Urology. 165 (2), 653 (2001).</li><li>Dmochowski, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+outlet+obstruction:+etiology+and+evaluation.+Reviews+in+Urology.">Bladder outlet obstruction: etiology and evaluation. Reviews in Urology.</a> 7 (Suppl 6), S3&#8211;S13. , (2005).</li><li>Aizawa, N., Homma, Y., Igawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+high+fat+diet+feeding+for+20+weeks+on+lower+urinary+tract+function+in+mice.">Influence of high fat diet feeding for 20 weeks on lower urinary tract function in mice.</a> Lower Urinary Tract Symptoms. 5 (2), 101 (2013).</li><li>Wang, Z., 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=Void+sorcerer:+an+open+source,+open+access+framework+for+mouse+uroflowmetry.">Void sorcerer: an open source, open access framework for mouse uroflowmetry.</a> American Journal of Clinical and Experimental Urology. 7 (3), 170 (2019).</li><li>Verstegen, A. M., Tish, M. M., Szczepanik, L. P., Zeidel, M. L., Geerling, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micturition+video+thermography+in+awake,+behaving+mice.">Micturition video thermography in awake, behaving mice.</a> Journal of Neuroscience Methods. 331, 108449 (2020).</li><li>Keller, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+urination+control+by+brainstem+neurons+that+relax+the+urethral+sphincter.">Voluntary urination control by brainstem neurons that relax the urethral sphincter.</a> Nature Neuroscience. 21 (9), (2018).</li><li>Hou, X. H., 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=Central+control+circuit+for+context-dependent+micturition.">Central control circuit for context-dependent micturition.</a> Cell. 167 (1), 73 (2016).</li><li>Negoro, H., 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=Involvement+of+urinary+bladder+Connexin43+and+the+circadian+clock+in+coordination+of+diurnal+micturition+rhythm.">Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm.</a> Nature Communication. 3, (2012).</li><li>Montalbetti, N., Carattino, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-sensing+ion+channels+modulate+bladder+nociception.">Acid-sensing ion channels modulate bladder nociception.</a> American Journal of Physiology. Renal Physiology. 321 (5), (2021).</li><li>Chen, H., Zhang, L., Hill, W. G., Yu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+voiding+spot+assay+in+mice:+a+simple+method+with+complex+environmental+interactions.">Evaluating the voiding spot assay in mice: a simple method with complex environmental interactions.</a> American Journal of Physiology. Renal Physiology. 313 (6), (2017).</li><li>Dalghi, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+distribution+of+PIEZO1+in+the+mouse+urinary+tract.">Expression and distribution of PIEZO1 in the mouse urinary tract.</a> American Journal of Physiology. Renal Physiology. 317 (2), 303 (2019).</li><li>Birder, L., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+modelling+of+interstitial+cystitis/bladder+pain+syndrome.">Animal modelling of interstitial cystitis/bladder pain syndrome.</a> International Neurourology Journal. 22, (2018).</li><li>Okinami, T., 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=Altered+detrusor+gap+junction+communications+induce+storage+symptoms+in+bladder+inflammation:+a+mouse+cyclophosphamide-induced+model+of+cystitis.">Altered detrusor gap junction communications induce storage symptoms in bladder inflammation: a mouse cyclophosphamide-induced model of cystitis.</a> PLoS One. 9 (8), (2014).</li><li>Tungtur, S. K., Nishimune, N., Radel, J., Nishimune, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Behavior+Tracker:+An+economical+method+for+tracking+behavior+in+home+cages.">Mouse Behavior Tracker: An economical method for tracking behavior in home cages.</a> Biotechniques. 63 (5), (2017).</li><li>Negoro, H., Kanematsu, A., Yoshimura, K., Ogawa, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronobiology+of+micturition:+putative+role+of+the+circadian+clock.">Chronobiology of micturition: putative role of the circadian clock.</a> The Journal of Urology. 190 (3), (2013).</li></ol>

The incorporation of video-monitoring is a cost-effective modification that presents several advantages over the classical VSA. In the classical VSA, which is typically used as an end-point assay, it is difficult to distinguish overlapping void spots. This is not a trivial concern, as mice tend to urinate multiple times in the same area when the assay is prolonged for several hours, typically in the corners of their cage. Thus, the first advantage of RT-VSA is that the investigator can readily identify individual spots t...

Urine storage and micturition are coordinated by a central circuitry (central nervous system) that receives information about the bladder filling status through the pelvic and hypogastric nerves. The urothelium, the epithelium that lines the urinary tract from the renal pelvis to the proximal urethra, forms a tight barrier to the metabolic waste products and pathogens present in urine. It is an integral component of a sensory web, which senses and communicates the filling state of the bladder to underlying tissues and afferent nerves1,2. Disruption of the urothelial barrier, or alterations in urothelial mechanotransduction pathways, can lead to voiding dysfunction along with lower urinary tract symptoms such as frequency, urgency, nocturia, and incontinence3,4,5,6,7. Likewise, aging, diabetes, lower urinary tract infections, interstitial cystitis, and other disease processes that affect the urinary bladder, or the associated circuitry that controls its function, are known to cause bladder dysfunction8,9,10,11,12,13,14,15,16,17,18,19. A better understanding of normal and abnormal voiding behavior depends on the development of methods that can reliably discriminate among different urination patterns.&#160;
Traditionally, the voluntary voiding behavior of mice has been studied using the void spot assay (VSA), developed by Desjardins and colleagues20, and broadly adopted due to its simplicity, low cost, and noninvasive approach8,21,22,23,24. This assay is typically performed as an endpoint assay, in which a mouse spends a defined amount of time in a cage lined by a filter paper, which is subsequently analyzed by counting the number and assessing the size of urine spots when the filter paper is placed under ultraviolet (UV) light (the urine spots fluoresce under these conditions)20. Despite these many advantages, the traditional VSA presents some major limitations. Because mice often urinate in the same areas, investigators have to restrict the duration of the assay to a relatively short period of time (&#8804;4 h)25. Even when the VSA is performed over shorter time periods, it is almost impossible to resolve small void spots (SVSs) that fall over large void spots or, to discriminate SVSs from the carryover of urine adhered to tails or paws. It is also very difficult to distinguish if SVSs are a consequence of frequent but individual voiding events (a phenotype that is often observed in response to cystitis4,26), or due to post-micturition dribbling (a phenotype associated with bladder outlet obstruction27). Furthermore, the desire to complete the assay during working hours, coupled with difficulties accessing housing facilities when the lights are turned off, often limits these assays to the light period of the 24 h circadian cycle. Thus, these time constraints prevent the evaluation of mouse voiding behavior during their active night phase, lessening the ability to analyze specific genes or treatments that are governed by circadian rhythms.&#160;
To overcome some of these limitations, researchers have developed alternative methods to assess voiding behavior in real time26,28,29,30,31,32. Some of these approaches involve the use of expensive equipment such as metabolic cages26,28,29, or the use of thermal cameras30; however, these too have limitations. For example, in metabolic cages, urine tends to adhere to the wires of the mesh floor and to the walls of the funnel, reducing the amount of urine that is collected and measured. Thus, it can be difficult to accurately collect data about small voids. Moreover, metabolic cages do not provide information about the spatial distribution of the voiding events (i.e., urination in the corners vs. the center of the chamber). Given that long-wavelength infrared radiation used by the thermographic cameras does not penetrate solids, voiding activity assessed by video thermography must be performed in an open system, which can be challenging with active mice, as they can jump several inches in the air. Another system is the automated voided stain on paper (aVSOP) approach33, which consists of rolled filter paper that winds up at a constant speed below the wire mesh floor of a mouse cage. This approach prevents paper damage and the overlap of urine spots that occur in the classical VSA, and its implementation allows the investigator to perform experiments over several days. However, it does not provide the investigator with precise timing of the voiding events, and there is no ability to examine behavior and how it correlates with spotting. To obtain this information, researchers have incorporated video-monitoring to voiding assays, an approach that allows the simultaneous assessment of mouse activity and urination events31,32. One approach consists of placing a blue light emitting diode (LED) and a video-camera with a green fluorescence protein filter set under the experimental cage to visualize the voiding events, and an infrared LED and a video-camera above the cage to capture mouse position32. This setup has been used to monitor voiding behavior while performing fiber photometry; however, the brightly lit environment of this system required the investigators to treat their mice with a diuretic agent to stimulate voiding. In another experimental design, wide-angle cameras were placed above and below the experimental cage to visualize mouse motor activity and urination events, respectively. In this case, urine spots deposited on a filter paper lining the cage&#8217;s floor were revealed by illuminating the filter paper with UV lights placed under the cage31. This setup was used in short assays, 4 min in duration, during the light phase of the day to study the brainstem neurons involved in voluntary voiding behavior31. The suitability of this system for its use during the dark phase or for periods of time &#62;4 min was not reported.&#160;
In this article, a method is described that enhances the traditional VSA by allowing for long-term video monitoring of mouse voiding behavior. This cost-effective approach provides temporal, spatial, and volumetric information about voiding events for extended periods of time during the light and dark phases of the day, along with details related to mouse behavior3,4,34. Detailed information for the construction of the voiding chambers, the implementation of a real-time VSA (RT-VSA), and the analysis of the data is provided. The RT-VSA is valuable for researchers seeking to understand the physiological mechanisms that control the function of the urinary system, to develop pharmacological approaches to control micturition, and to define the molecular basis of disease processes that affect the lower urinary tract.&#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 10 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 20</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 12 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 6</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 40 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 4</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211; cut to 14.75 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 12</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211; cut to 32 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 5</td>
		</tr>
		<tr>
			<td>1/4-20 Double Slide-in Economy T-Nut</td>
			<td>80/20</td>
			<td>3280</td>
			<td>Amount: 16</td>
		</tr>
		<tr>
			<td>1/4-20 Triple Slide-in Economy T-Nut</td>
			<td>80/20</td>
			<td>3287</td>
			<td>Amount: 18</td>
		</tr>
		<tr>
			<td>10 &#38; 25 Series 2 Hole - 18mm Slotted Inside Corner Bracket with Dual Support</td>
			<td>80/20</td>
			<td>14061</td>
			<td>Amount: 6</td>
		</tr>
		<tr>
			<td>10 Series 3 Hole - Straight Flat Plate</td>
			<td>80/20</td>
			<td>4118</td>
			<td>Amount: 8</td>
		</tr>
		<tr>
			<td>10 Series 5 Hole - &#34;L&#34; Flat Plate</td>
			<td>80/20</td>
			<td>4081</td>
			<td>Amount: 8</td>
		</tr>
		<tr>
			<td>10 Series 5 Hole - &#34;T&#34; Flat Plate</td>
			<td>80/20</td>
			<td>4080</td>
			<td>Amount: 8</td>
		</tr>
		<tr>
			<td>10 Series 5 Hole - Tee Flat Plate</td>
			<td>80/20</td>
			<td>4140</td>
			<td>Amount: 2</td>
		</tr>
		<tr>
			<td>10 Series Standard Lift-Off Hinge - Right Hand Assembly</td>
			<td>80/20</td>
			<td>2064</td>
			<td>Amount: 2</td>
		</tr>
		<tr>
			<td>10 to 15 Series 2 Hole - Lite Transition Inside Corner Bracket</td>
			<td>80/20</td>
			<td>4509</td>
			<td>Amount: 6</td>
		</tr>
		<tr>
			<td>24&#8221;-long UV tube lights</td>
			<td>ADJ Products LLC</td>
			<td>T8-F20BLB24</td>
			<td>Amount: 2 
			20W bulb &#8211; 24&#8221; Wavelength: 365nm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 1 
			82.5 cm x 26.5 cm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 2 
			26.5 cm X 30.5 cm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 2 
			82.5 cm x 30.5 cm</td>
		</tr>
		<tr>
			<td>AR polycarbonate (UV resistance)</td>
			<td>80/20</td>
			<td>65-2641</td>
			<td>Amount: 2 
			4.5mm Thick, Clear, 38.5 cm x 26.5 cm</td>
		</tr>
		<tr>
			<td>AR polycarbonate (UV resistance)</td>
			<td>80/20</td>
			<td>65-2641</td>
			<td>Amount: 4 
			4.5mm Thick, Clear, 38.5 cm x 21.5 cm</td>
		</tr>
		<tr>
			<td>AR polycarbonate (UV resistance)</td>
			<td>80/20</td>
			<td>65-2641</td>
			<td>Amount: 4 
			4.5mm Thick, Clear, 26.5 cm x 21.5 cm</td>
		</tr>
		<tr>
			<td>AR polycarbonate (UV resistance)</td>
			<td>80/20</td>
			<td>65-2641</td>
			<td>Amount: 4 
			4.5mm Thick, Clear 37.5 cm x 23.9 cm</td>
		</tr>
		<tr>
			<td>AR polycarbonate (UV resistance)</td>
			<td>80/20</td>
			<td>65-2641</td>
			<td>Amount: 4 
			4.5mm Thick, Clear , 24.4 cm x 23.9 cm</td>
		</tr>
		<tr>
			<td>Chromatography paper (thin paper)</td>
			<td>&#160;Thermo Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmos blotting paper (thick paper)</td>
			<td>Blick Art Materials</td>
			<td>10422-1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Version 9.4.0</td>
			<td>graphing and statistics software</td>
		</tr>
		<tr>
			<td>ImageJ FIJI</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Merck</td>
			<td></td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Quick Time Player 10.5 software&#160;</td>
			<td>Apple</td>
			<td></td>
			<td>multimedia player</td>
		</tr>
		<tr>
			<td>Security spy</td>
			<td>Ben software</td>
			<td></td>
			<td>video surveillance software system</td>
		</tr>
		<tr>
			<td>Standard End Fastener, 1/4-20</td>
			<td>80/20</td>
			<td>3381</td>
			<td>Amount: 80</td>
		</tr>
		<tr>
			<td>UV transmitting acrylic</td>
			<td>Spartech</td>
			<td>Polycast Solacryl SUVT</td>
			<td>Amount: 2 
			38.5 cm x 26.5 cm&#160;</td>
		</tr>
		<tr>
			<td>Water gel: HydroGel</td>
			<td>ClearH2O&#160;</td>
			<td>&#160;70-01-5022</td>
			<td>(https://www.clearh2o.com/product/hydrogel/)</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C930e</td>
			<td>Amount: 4</td>
		</tr>
	</tbody>
</table>

real-time void spot assay

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the nervous system. To study voluntary voiding behavior in mouse models, researchers have developed the void spot assay (VSA), a method that measures the number and area of urine spots deposited on a filter paper lining the floor of an animal&#8217;s cage. Although technically simple and inexpensive, this assay has limitations when used as an end-point assay, including a lack of temporal resolution of voiding events and difficulties quantifying overlapping urine spots. To overcome these limitations, we developed a video-monitored VSA, which we call real-time VSA (RT-VSA), and which allows us to determine voiding frequency, assess voided volume and voiding patterns, and make measurements over 6 h time windows during both the dark and light phases of the day. The method described in this report can be applied to a wide variety of mouse-based studies that explore the physiological and neurobehavioral aspects of voluntary micturition in health and disease states.

Voiding behavior of urothelial Piezo1/2 knockout mice
During the storage phase of the micturition cycle, the urothelium is hypothesized to sense the tension exerted by the urine accumulated in the bladder and to transduce this mechanical stimulus into cellular responses such as serosal ATP release1,3. We have previously shown that mechanically activated PIEZO1 and PIEZO2 channels are expressed in the mouse uro...

Watch this Scientific Journal Video about Real-Time Void Spot Assay at JoVE.com

Real-Time Void Spot Assay

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the ...

Research

JoVE Journal

Behavior

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for ...

Brain State-dependent Brain Stimulation with Real-time Electroencephalography-Triggered Transcranial Magnetic Stimulation

The effect of a stimulus to the brain depends not only on the parameters of the stimulus but also on the dynamics of brain activity at the time of the ...

A Cross-Disciplinary and Multi-Modal Experimental Design for Studying Near-Real-Time Authentic Examination Experiences

Over the past ten years, research into students&#39; emotions in educational environments has increased. Although researchers have called for more studies ...

Implementation of a Real-Time Psychosis Risk Detection and Alerting System Based on Electronic Health Records using CogStack

Recent studies have shown that an automated, lifespan-inclusive, transdiagnostic, and clinically based, individualized risk calculator provides a powerful ...

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Real-Time Proxy-Control of Re-Parameterized Peripheral Signals using a Close-Loop Interface

The fields that develop methods for sensory substitution and sensory augmentation have aimed to control external goals using signals from the central ...

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates ...