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.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
In This Article
Summary
Abstract
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’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.
Introduction
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 mechan....
Access restricted. Please log in or start a trial to view this content.
Protocol
Urothelial 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 (1.5–3 months old and 17–20 g in weight) and male (2–4 months old and 23–29 g in weight) mice were used in the experiments. For cyclophosphamide....
Access restricted. Please log in or start a trial to view this content.
Results
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.......
Access restricted. Please log in or start a trial to view this content.
Discussion
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.......
Access restricted. Please log in or start a trial to view this content.
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
....Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1.00” X 1.00” T-Slotted Profile - Four Open T-Slots – cut to 10 inches | 80/20 | 1010 | Amount: 20 |
| 1.00” X 1.00” T-Slotted Profile - Four Open T-Slots – cut to 12 inches | 80/20 | 1010 | Amount: 6 |
| 1.00” X 1.00” T-Slotted Profile - Four Open T-Slots – cut to 40 inches | 80/20 | 1010 | Amount: 4 |
| 1.00” X 1.00” T-Slotted Profile - Four Open T-Slots – cut to 14.75 inches | 80/20 | 1010 | Amount: 12 |
| 1.00” X 1.00” T-Slotted Profile - Four Open T-Slots – cut to 32 inches | 80/20 | 1010 | Amount: 5 |
| 1/4-20 Double Slide-in Economy T-Nut | 80/20 | 3280 | Amount: 16 |
| 1/4-20 Triple Slide-in Economy T-Nut | 80/20 | 3287 | Amount: 18 |
| 10 & 25 Series 2 Hole - 18mm Slotted Inside Corner Bracket with Dual Support | 80/20 | 14061 | Amount: 6 |
| 10 Series 3 Hole - Straight Flat Plate | 80/20 | 4118 | Amount: 8 |
| 10 Series 5 Hole - "L" Flat Plate | 80/20 | 4081 | Amount: 8 |
| 10 Series 5 Hole - "T" Flat Plate | 80/20 | 4080 | Amount: 8 |
| 10 Series 5 Hole - Tee Flat Plate | 80/20 | 4140 | Amount: 2 |
| 10 Series Standard Lift-Off Hinge - Right Hand Assembly | 80/20 | 2064 | Amount: 2 |
| 10 to 15 Series 2 Hole - Lite Transition Inside Corner Bracket | 80/20 | 4509 | Amount: 6 |
| 24”-long UV tube lights | ADJ Products LLC | T8-F20BLB24 | Amount: 2 20W bulb – 24” Wavelength: 365nm |
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 1 82.5 cm x 26.5 cm | |
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 2 26.5 cm X 30.5 cm | |
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 2 82.5 cm x 30.5 cm | |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 2 4.5mm Thick, Clear, 38.5 cm x 26.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear, 38.5 cm x 21.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear, 26.5 cm x 21.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear 37.5 cm x 23.9 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear , 24.4 cm x 23.9 cm |
| Chromatography paper (thin paper) | Thermo Fisher Scientific | 57144 | |
| Cosmos blotting paper (thick paper) | Blick Art Materials | 10422-1005 | |
| Excel | Microsoft Corporation | ||
| GraphPad Prism | GraphPad Software | Version 9.4.0 | graphing and statistics software |
| ImageJ FIJI | NIH | ||
| Parafilm | Merck | transparent film | |
| Quick Time Player 10.5 software | Apple | multimedia player | |
| Security spy | Ben software | video surveillance software system | |
| Standard End Fastener, 1/4-20 | 80/20 | 3381 | Amount: 80 |
| UV transmitting acrylic | Spartech | Polycast Solacryl SUVT | Amount: 2 38.5 cm x 26.5 cm |
| Water gel: HydroGel | ClearH2O | 70-01-5022 | (https://www.clearh2o.com/product/hydrogel/) |
| Webcam | Logitech | C930e | Amount: 4 |
References
- Dalghi, M. G., Montalbetti, N., Carattino, M. D., Apodaca, G. The urothelium: life in a liquid environment. Physiological Reviews. 100 (4), 1621(2020).
- de Groat, W. C., Griffiths, D., Yoshimura, N. Neural control of the lower urinary tract. Comprehensive Physiology. 5 (1), 327(2....
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
