Real-Time Void Spot Assay

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.