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Voluntary urination control by brainstem neurons that relax the urethral sphincter

Nature Neuroscience volume 21, pages 1229–1238 (2018)Cite this article

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Abstract

Voluntary urination ensures that waste is eliminated when safe and socially appropriate, even without a pressing urge. Uncontrolled urination, or incontinence, is a common problem with few treatment options. Normal urine release requires a small region in the brainstem known as Barrington’s nucleus (Bar), but specific neurons that relax the urethral sphincter and enable urine flow are unknown. Here we identify a small subset of Bar neurons that control the urethral sphincter in mice. These excitatory neurons express estrogen receptor 1 (BarESR1), project to sphincter-relaxing interneurons in the spinal cord and are active during natural urination. Optogenetic stimulation of BarESR1 neurons rapidly initiates sphincter bursting and efficient voiding in anesthetized and behaving animals. Conversely, optogenetic and chemogenetic inhibition reveals their necessity in motivated urination behavior. The identification of these cells provides an expanded model for the control of urination and its dysfunction.

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Fig. 1: A novel cell type in Barrington’s nucleus with projections biased to sphincter-inhibiting interneurons.
Fig. 2: BarESR1 activity increases during urination events.
Fig. 3: Photostimulation of BarESR1 neurons induces efficient urination in awake and anesthetized animals.
Fig. 4: BarESR1 neurons facilitate urine release during cystometry.
Fig. 5: BarESR1 neurons relax the urethral sphincter by promoting bursting activity.
Fig. 6: Naive male mice rapidly and robustly scent mark to female odor cues.
Fig. 7: Chemogenetic and optogenetic inhibition of BarESR1 neurons impairs voluntary scent marking urination.

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Acknowledgements

We thank W. de Groat for constructive comments and discussions on an earlier version of the manuscript and S. Mukhopadhyay for help with behavior. J.A.K. was supported by NSF-GRFP grant DGE-1144086. L.S is funded by NSF IOS-1556085 and NIH R01-DC015253.

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Authors and Affiliations

  1. Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, CA, USA

    Jason A. Keller, Jingyi Chen, Sierra Simpson, Olivier George & Lisa Stowers

  2. Neurosciences Graduate Program, University of California San Diego, La Jolla, CA, USA

    Jason A. Keller

  3. Biomedical Sciences Graduate Program, The Scripps Research Institute, La Jolla, CA, USA

    Jingyi Chen & Sierra Simpson

  4. Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA

    Eric Hou-Jen Wang & Varoth Lilascharoen

  5. Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

    Byung Kook Lim

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Contributions

J.A.K., J.C. and L.S. designed the study, analyzed the data and wrote the manuscript. S.S. aided in the cystometry; O.G. supported S.S. E.H.-J.W and B.K.L. aided in the fiber photometry. V.L. performed slice physiology. All other experiments were performed by J.A.K. and J.C.

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Correspondence to Lisa Stowers.

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Integrated supplementary information

Supplementary Figure 1 Neurotransmitter identity and direct spinal projections of BarESR1 neurons.

a, Anti-ESR1 overlap with ESR1-ZsGreen (Ai6) genetic reporter. Bottom is larger view without Nissl. b, Anti-ESR1 overlap with Vglut2-ZsGreen genetic reporter. c, Anti-ESR1 overlap with Vgat-ZsGreen genetic reporter. d, RnaScope in-situ hybridization of Crh/Esr1/Vglut2 mRNA in Bar region of a wild-type male mouse, 20X objective. e, Larger view of dotted area in (d), 40X objective. f, g, Close-up views of individual cells in (e), with DAPI counterstain. h-k, same as (d)-(g), but with Vgat mRNA probe. l, Schematic of CTB injection into S1 spinal cord. m, CTB injection site. n, Retrograde CTB labeling in Bar with anti-ESR1 and CRH-tdT. Dotted ovals delineate Bar. Scale bars = 100 μm, except panels f/g/j/k, scale bars = 20 μm.

Supplementary Figure 2 BarESR1 and BarCRH projections to urinary nuclei in the spinal cord.

a, Schematic for testing Bar cell type axonal projections to spinal cord. b, Representative BarCRH axon projections at the L1/L4/L6/S2 spinal cord levels, separate from example in Fig. 1g-i. Top is ArchT virus, bottom is ChR2 virus. c, Same as (b), but for BarESR1 axon projections. Scale bars = 100 μm.

Supplementary Figure 3 Visualizing and quantifying urination behavior.

a, Schematic of behavioral setup for simultaneous optogenetics/fiber photometry, video recording, and analysis of urine excretion of awake behaving mice. b, Left: top camera records mouse position, right: urine fluoresces under UV light enabling excretion to be visualized throughout assay. Grey carrot indicates position between synchronized images from top and bottom cameras. c, Example of automated urine pixel detection with calibration data consisting of 4 replicates of 8 different volumes of male mouse urine on thin chromatography paper. d, Second order polynomial fit to calibration data on thick and thin paper; coefficients from these were used to calculate all urine amounts reported in microliters.

Supplementary Figure 4 Whole-cell recordings of BarCRH-ChR2 and BarESR1-ChR2 neurons during photostimulation.

a, Example current clamp traces from representative BarCRH-ChR2 (magenta), BarESR1-ChR2 (green), and BarESR1-GFP (orange) neurons during 5 sec photostimulation bouts at 10/25/50 Hz. b, Visualization of recording location of BarESR1-ChR2 neuron in (a) showing ChR2-tdTomato expression. c, Stimulated firing rates (each circle represents the mean firing rate during the stimulation period for an individual neuron) versus photostimulation frequency for all recorded BarCRH-ChR2 (magenta, n = 6), BarESR1-ChR2 (green, n = 12), and BarESR1-GFP (orange, n = 4) neurons. Most neurons, particularly BarCRH-ChR2, are affected by depolarization block at 50 Hz. Scale bars = 100 μm.

Supplementary Figure 5 Frequency characteristics of urethral sphincter bursting during natural behavior and after Bar photostimulation.

a, Schematic for optogenetic Bar stimulation during EUS EMG recording. b, Example raw EUS EMG and corresponding spectral power in the 5-15 Hz band for photostimulated burst responses in BarCRH-ChR2 (top) and BarESR1-ChR2 (bottom) mice. BarCRH-ChR2 burst is preceded by an increase in tonic activity during the photostimulation period, whereas BarESR1-ChR2 burst occurs at low latency without preceding tonic activity, and displays decreasing frequency characteristic of natural bursts in (f). c, Example video frame from wireless corpus spongiosum pressure recording in the presence of female odor (yellow shading). d, Schematic of corpus spongiosum recording setup. e, Corpus spongiosum pressure recording after presentation of female odor. Top, raw pressure; bottom, spectral power in the 5-15 Hz band. Yellow arrows mark approximate start times for urination events. f, Shorter timescale recordings as in (e), for 5 urination events across 2 mice. Binary images on the left show relative sizes of thresholded urine marks corresponding to bursts on the right. Frequency typically decreases over a large burst lasting a few seconds, although shorter bursts with less urine were also observed (2nd from top).

Supplementary Figure 6 BarESR1-ChR2 photostimulation can enable urethral sphincter relaxation without bladder contraction.

a, Three example BarESR1-ChR2 photostimulation trials in the empty bladder condition (from heatmap in Fig. 5b), in which burst-like EMG activity was observed in the absence of bladder response. Top/yellow traces are bladder pressure, bottom/red traces are raw EMG. Blue shading and dotted lines delineate photostimulation periods.

Supplementary Figure 7 Behavioral controls for BarESR1-hM4Di and BarCRH-hM4Di chemogenetic inhibition.

a, Total distance traveled during the assay shown in Fig. 7a-e for BarESR1-hM4Di mice (n = 8). Thin dotted lines are individuals, thick black line is mean ± s.e.m. p = 0.58 Friedman’s test. b, Same as (b), but for total female urine odor sniffing time. p = 0.021 Friedman’s test, *day 4 saline p = 0.012 Dunn-Sidak posthoc differences from CNO day 1. c, Analysis of BarCRH-hM4Di mice (n = 10) injected with either saline or CNO on consecutive days prior to a 2-hour urination assay, similar to that previously published15 and which is not limited to odor-evoked, voluntary urination. *p = 0.049, Mann-Whitney U test for difference between CNO and saline treatment days.

Supplementary Figure 8 BarESR1-ArchT photoinhibition terminates sphincter bursting during cystometry and does not result in rebound urination in awake mice.

a, Example ArchT-GFP expression in Bar of ESR1-Cre mouse; right, larger views minus Nissl. b, Number of Bar cells infected with ArchT virus versus total urine with light OFF (not inhibited) divided by that with light on (while inhibited) for all mice (n = 3). c, Δurine amount around two 30 second photoinhibition periods followed by one 2 min. photoinhibition period, during which control odor only was present. n = 9 total photoinhibition bouts from 3 mice. d, Bladder pressure and sphincter EMG during cystometry, top, with natural unimpeded cycling, and bottom, in which 2 seconds of BarESR1-ArchT photoinhibition was triggered as soon as filling evoked bursting was detected, which terminates bursting within ~100 ms, often followed by tonic contractions such as with cessation of ChR2 photostimulation. e, Heatmap of mean EMG power density at bursting frequencies (5-15 Hz) during BarESR1-ArchT photoinhibition as in bottom of panel d (top: 2 second inhibition trials; bottom: 5 second inhibition trials; n = 2 mice). Green shading or red dotted lines mark photoinhibition periods. Scale bars = 100 μm.

Supplementary Figure 9 Simplified summary of a nose-to-sphincter circuit and other potential Bar functions.

a, BarESR1 (green) and BarCRH (magenta) neurons are intermingled (cell overlay from Fig. 1d), and the minority BarESR1 population projects to both the mediolateral column (ML) and heavier to the dorsal grey commissure (DGC), which directly inhibits sphincter motoneurons in the dorsolateral nucleus (DL). Activation of BarESR1 neurons increases bladder pressure and simultaneously inhibits the sphincter via bursting, thus driving efficient urine excretion, whereas activation of BarCRH neurons produces a focal increase in bladder pressure and either no effect at the sphincter or a tonic excitation resembling the guarding reflex. Thoracolumbar projections of BarESR1 neurons are not shown, as well as afferent feedback connections from bladder and urethra. b, Many factors influence a variety of pelvic functions, and heterogeneity in Bar may allow differential coordination of both somatic and autonomic targets for various behaviors.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9

Reporting Summary

Supplementary Video 1

Awake photostimulation of BarESR1-ChR2 neurons at five different frequencies. This video shows a freely moving BarESR1-ChR2 mouse urinating in response to light pulses at five frequencies: 1, 5, 10, 25, 50 Hz. Photostimulation occurs when white frequency letters appear on middle-left. Top: recording from top camera (to visualize subject movement), Bottom: recording from bottom camera (to visualize and quantify urination). For schematic of assay chamber see Supplementary Fig. 3. Video condensed to 4× speed.

Supplementary Video 2

Awake photostimulation of BarCRH-ChR2 neurons at five different frequencies. This video shows a freely moving BarCRH-ChR2 mouse urinating in response to light pulses at five frequencies: 1, 5, 10, 25, 50 Hz. Photostimulation occurs when white frequency letters appear on middle-left. This animal underwent subsequent cystometry and showed reliable bladder pressure increases in response to photostimulation, as shown in Fig. 4c (bottom). Video condensed to 4× speed in a two-camera recording set up as in Supplementary Video 1.

Supplementary Video 3

Anesthetized photostimulation of BarESR1-ChR2 neurons. This video shows an anesthetized BarESR1-ChR2 mouse urinating in response to three 50 Hz photostimulation bouts separated by 1 min intervals. The subject is moved between stimulations to observe and record urine excreted during each photoperiod. Video condensed to 4× speed in a two-camera recording set up as in Supplementary Video 1.

Supplementary Video 4

Photostimulation of BarESR1-ChR2neurons during cystometry. This video shows urine output during BarCRH-ChR2 photostimulation while recording bladder pressure (top yellow rolling trace) and urethral sphincter EMG (bottom red rolling trace). Blue shading on plots and blue light shadow behind mouse delineate photostimulation period. Shorter sphincter bursting and urine excretion does not occur until after photostimulation. Video slowed to 0.67× speed to show bursting clearly.

Supplementary Video 5

Photostimulation of BarCRH-ChR2neurons during cystometry. This video shows urine output during BarESR1-ChR2 photostimulation while recording bladder pressure (top yellow rolling trace) and urethral sphincter EMG (bottom red rolling trace). Blue shading on plots and blue light shadow behind mouse delineate photostimulation period.

Supplementary Video 6

Odor-motivated urination assay. This video shows example urine marking behavior under UV light in response to control odor (1.5–2 min) and female odor (2–4 min) in a 4 min assay. Video condensed to 4× speed in a two-camera recording set up as in Supplementary Video 1.

Supplementary Video 7

Photoinhibition of BarESR1-ArchTneurons prevents rapid, odor-evoked urination. This video shows a BarESR1-ArchT mouse before, during, and after a 2 min photoinhibition period during which female urine was presented. Video condensed to 4× speed in a two-camera recording set up as in Supplementary Video 1.

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Keller, J.A., Chen, J., Simpson, S. et al. Voluntary urination control by brainstem neurons that relax the urethral sphincter. Nat Neurosci 21, 1229–1238 (2018). https://doi.org/10.1038/s41593-018-0204-3

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