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>
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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. 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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;

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		<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 ...

The method described in this report can be used to understand physiological and neuro-behavioral aspects of voluntary micturition in health and disease states. This technique allows us to monitor mouse volume behavior in the light and dark phases of the day, and provides temporal, spatial, and volumetric information on voiding events. To prepare a real-time void spot assay or RT-VSA recording chamber, place a thin or thick filter paper, depending on the time of day, at the bottom of the RT-VSA recording cage.On top of the filter paper, place a plastic igloo for sleeping space;a sterile 1.5 ml microcentrifuge tube for enrichment purposes;and a 60 mm x 15 mm plastic dish containing two or three pieces of mouse dry chow;and 14 to 16 grams of water in the form of a gel pack. Once the recording chamber is ready, gently placed the mouse on the filter paper. Ensure that the transfer of the mouse from the housing cage to the recording cage occurs with minimum stress.Once the mouse is inside the recording cage, place the lid and cover the top of the lid with an absorbent bench blue pad to minimize direct ambient light reflections on the plexiglass lid surface. Turn on the ultraviolet lights in the lower chamber. To record RT-VSA video from the top and bottom cameras, use video surveillance recording software configured to record simultaneously from multiple webcams or networked cameras.Initiate recording by pressing Cmd R in the program window. Perform video recordings at one frame per second. Immediately after initiating the recordings, exit the room and close the door gently.Ensure that the room remains quiet for the entire experiment. Stop the recordings by pressing Cmd T.Turn off the ultraviolet lights. After stopping the recording, the software automatically generates a movie file in m4v format for each camera and saves it under the camera's name in a previously selected destination folder.Verify that within each camera's folder the experiments are organized into folders by date. In each date or experiment folder, verify that there is one m4v file and all of the individual jpeg files that correspond to each movie frame. Create a folder on the desktop with the name and date of the experiment and transfer the m4v files into this folder.Copy the movie folder into a flash drive for analysis on an external computer. Open a movie file collected by the bottom camera during the light phase or the upper camera during the dark phase for analysis. After assessing the quality of the movie, analyze experiments by moving to the correct time window using the fast forward command or the time bar slider.Voiding activity during the light phase is recorded between 11:00 AM and 5:00 PM, and during the dark phase, between midnight and 6:00 AM.Play the movie in fast forward mode by clicking on the fast forward icon or manually scroll through the movie, looking for evidence that the mouse is voiding. Look for the sudden appearance of bright urine spots on the filter paper or the behavioral changes, including movement to the corners of the cage and a brief period of inactivity when the mouse is voiding. Register the time at which each void occurs.As a convention, the time of the void is recorded at the first sign the urine is detected. To take measurements of the void, first use the scroll bar to move forward or backward in time, looking for the point when maximal diffusion of the urine spot has occurred. Pause the movie at this point and place the computer mouse arrow at the spot under analysis to mark the spot of interest in the screenshot.Name the screenshot file using correlative numbers to account for the order of appearance in the movie. Once all the void spots have been analyzed, measure the total area of the filter paper by capturing a screenshot and delineating the border of the filter paper. Calculate the percentage area of each of the urine spots.Transform the values of the percentage area into the volume of urine for each void spot using the calibration curves and the interpolate function in the graphing software. The voiding behavior of female and male Piezo1, 2, knockout and control mice were recorded during the dark and light phase of the day. During their inactive light phase, no significant differences were observed in any parameters analyzed for female or male knockout mice compared to the control.However, in the active dark phase, female and male knockout mice showed altered voiding phenotype, characterized by a significant increase in the number and total volume of small void spots. There was no significant differences in primary voids spots or PVS number, average volume per PVS, or total PVS volume in female or male knockout mice compared to the control mice. The voiding behavior of female mice was tested under basal conditions and following cyclophosphamide treatment.Compared to the basal conditions, the amount of urine release per void is smaller after cyclophosphamide treatment and the micturition events are much more frequent. One key aspect for assay reproducibility is to manipulate the animals as gently as possible and keep them under minimum stress during the whole test period. As this is an noninvasive assay, additional procedures can be performed afterward including cytometry and electromyography to assess bladder and external urethra sphincter functions respectively.Implementing this technique will allow researchers to define the contribution of specific pathways to voiding behavior in health and disease states.

Research

JoVE Journal

Behavior

1.7K visualizações.  University of Pittsburgh. O método descrito neste relato pode ser utilizado para compreender aspectos fisiológicos e neurocomportamentais da micção voluntária em estados de saúde e doença. Essa técnica permite monitorar o comportamento do volume do mouse nas fases clara e escura do dia, além de fornecer informações temporais, espaciais e volumétricas sobre eventos miccionais. Para preparar um ensaio de ponto vazio em tempo real ou uma câmara de gravação RT-VSA, coloque um papel de filtro fino ou grosso,...