Afferent nerve activity in a mouse model increases with faster bladder filling rates in vitro, but voiding behavior remains unaltered in vivo

Abstract

Storage and voiding functions in urinary bladder are well-known, yet fundamental physiological events coordinating these behaviors remain elusive. We sought to understand how voiding function is influenced by the rate at which the bladder fills. We hypothesized that faster filling rates would increase afferent sensory activity and increase micturition rate. In vivo, this would mean animals experiencing faster bladder filling would void more frequently with smaller void volumes. To test this hypothesis, we measured afferent nerve activity during different filling rates using an ex vivo mouse bladder preparation and assessed voiding frequency in normally behaving mice noninvasively (UroVoid). Bladder afferent nerve activity depended on the filling rate, with faster filling increasing afferent nerve activity at a given volume. Voiding behavior in vivo was measured in UroVoid cages. Male and female mice were given access to tap water or, to induce faster bladder filling rates, water containing 5% sucrose. Fluid intake increased dramatically in mice consuming 5% sucrose. As expected, micturition frequency was elevated in the sucrose group. However, even with the greatly increased rate of urine production, void volumes were unchanged in both genders. Although faster filling rates generated higher afferent nerve rates ex vivo, this did not translate into more frequent, smaller-volume voids in vivo. This suggests afferent nerve activity is only one factor contributing to the switch from bladder filling to micturition. Together with afferent nerve activity, higher centers in the central nervous system and the state of arousal are likely critical to coordinating the micturition reflex.

Keywords: incontinence; micturition; overactive bladder; polydipsia; polyuria.

Figure 1.

Afferent nerve activity increases with faster bladder filling rates ex vivo. Original recordings of bladder pressure (A) and afferent nerve frequency (B) in a representative, isolated, cannulated mouse urinary bladder subjected to filling with saline at rates of 0.2, 0.6, and 1.8 mL/h, in descending fashion. At each rate, bladder was filled until intravesical pressure reached 25 mmHg, at which time the filling was stopped and the bladder was emptied. C: maximum afferent nerve frequency reached during filling at each of three filling rates tested. Points represent individual bladder measurements (n = 8), and horizontal lines indicate means ± SE. *P < 0.05 vs. 0.2 mL/h. †P < 0.05 vs. 0.6 mL/h. D: volume reached at the end of each filling (Max Bladder Capacity). Points represent individual bladder measurements (n = 8), and horizontal lines indicate means ± SE. There were no significant differences. For C and D, a one-way repeated measures ANOVA followed by Tukey’s test for multiple comparisons was used.

Figure 2.

Maximal afferent nerve frequency reached during filling increased at faster filling rates, but the sensitivity of afferent nerves did not change. A: absolute afferent nerve activity plotted against normalized bladder capacity (see text for description) at each filling rate (n = 8 bladders). *P < 0.05 for all filling rates. B: normalized afferent nerve activity (%Max) expressed as a function of normalized bladder capacity at each filling rate (n = 8 bladders). Filling rate did not significantly affect normalized nerve activity relative to the fractional filling state. C: absolute afferent nerve activity plotted against bladder pressure in 2 mmHg bins. *P < 0.05 for all filling rates. Data were analyzed using a two-way repeated measures ANOVA followed by Tukey’s test for multiple comparisons.

Figure 3.

Original voiding micturograms from mice ingesting tap water (A and C) and 5% sucrose (B and D). A and B: raw data from the analytical balance that weighed each void event. Baseline (green dots) and peak (red dot) for each void is indicated. Shaded rectangles indicate the dark phase of the light:dark cycle. C and D: void volumes for every void extracted from raw data, plotted as individual void volumes (blue dots/lines, left Y-axis), or as a cumulative record with each void volume added to the prior void volume (orange lines, right Y-axis). Shaded rectangles indicate the dark phase of the light:dark cycle. E: total number of voids during the 48-h voiding study observed in mice consuming tap water (n = 7 mice) or 5% sucrose (n = 8 mice). F: average intermicturition interval (IMI) for all void events from each animal recorded during the 48-h voiding study. E and F, individual subject data are plotted as circles, and horizontal lines indicate means ± SE. *P < 0.05 vs. tap water. An unpaired Mann–Whitney test on ranks was used for statistical analysis.

Figure 4.

A: total water intake during the 48-h voiding study for mice consuming tap water (open circles, n = 7 mice) or 5% sucrose (closed circles, n = 8 mice). B: total void volume during the 48-h voiding study. C: correlation plot showing total void volume plotted against total water intake for all individual subjects. D: average void volume obtained by averaging the volume of all individual void events detected in each subject. E: average urine production rate (UPR) obtained by averaging all individual void events from each subject. In all panels, individual subject data are plotted as circles, and horizontal lines indicate means ± SE. *P < 0.05 vs. tap water. An unpaired Mann–Whitney test on ranks was used for all statistical analyses.

Figure 5.

A: number of voids detected during 3-h time bins in mice consuming tap water (open circles, n = 7 mice) or 5% sucrose (closed circles, n = 8 mice). B: average void volumes recorded during 3-h time bins in mice consuming tap water or 5% sucrose. Shaded rectangles indicate dark portion of the light:dark cycle. *P < 0.05 between the tap water and sucrose groups. NS, no significant difference between tap water and sucrose groups. A two-way repeated measures ANOVA with Greenhouse–Geisser correction for unequal variances was used for all statistical comparisons.

Figure 6.

Seventy-two hour voiding behavior in male mice (n = 8 mice). A: number of voids measured in 3-h time bins during the 72-h voiding study in which mice had access to tap water for the first and third 24-h periods and 5% sucrose water during the second 24-h period. Shaded rectangles indicate the dark portion of the light:dark cycle. B: average intermicturition interval (IMI) detected in 3-h time bins during the 72-h voiding study. C: average number of voids detected in the light and dark phases of the light:dark cycle in mice drinking tap water (open circles) and 5% sucrose (closed circles). D: average IMI detected in light and dark phases of the light:dark cycle. E: average void volumes measured in light and dark phases of the light:dark cycle. For C–E, voiding behavior was quantified during the final 6 h of light/dark cycles. Individual subject data are plotted as circles, and horizontal lines are means ± SE. *P < 0.05 vs. tap water light cycle. †P < 0.05 vs. sucrose light cycle. ‡P < 0.05 vs. tap water dark cycle. A two-way repeated measures ANOVA with Tukey’s test for multiple comparisons was used for all statistical analyses.

Figure 7.

Seventy-two hour voiding behavior in female mice (n = 8 mice). A: original voiding micturogram obtained from a female mouse during the 72-h voiding study in which mice had access to tap water for the first and third 24-h periods and 5% sucrose water during the second 24-h period. Shaded rectangles indicate the dark portion of the light:dark cycle. B: average number of voids detected in the light and dark phases of the light:dark cycle in mice drinking tap water (open circles) and 5% sucrose (closed circles). C: average IMI detected in light and dark phases of the light:dark cycle. D: average void volumes measured in light and dark phases of the light:dark cycle. For B–D, voiding behavior was quantified during the final 6 h of the light:dark cycles. Individual subject data are plotted as circles, and horizontal lines are means ± SE. *P < 0.05 vs. tap water light cycle. †P < 0.05 vs. sucrose light cycle. ‡P < 0.05 vs. tap water dark cycle. A two-way repeated measures ANOVA with Tukey’s test for multiple comparisons was used for all statistical analyses. IMI, intermicturition interval.