The activity of PMC cells tightly correlates with bladder contraction and sphincter relaxation during successful voiding.

(A) Schematic (top) and representative histology (bottom) of optrode recording in the PMC of an ESR1-cre mouse. Scale bar: 200 µm. (B) Cumulative sessions of sorted single-unit activity of PMCESR1+ (upper; n = 28 cells from 4 mice) and non-PMCESR1+ cells (lower; n = 51 cells from 4 mice) aligned to voiding onset (black dashed line), vertically arranged by their instantaneous firing rate at the voiding onset. (C) Boxplots showing the baseline-corrected average firing rates before and during voiding among PMCESR1+ (top, n = 28 cells from 4 mice, **P = 0.004) and non-PMCESR1+ cells (bottom, n = 51 cells from 4 mice, P = 0.6; n.s., not significant; Wilcoxon signed-rank test). (D) Schematic of fiber photometry recording for PMCESR1+ cells during simultaneous cystometry and urethral electromyography in a urethane-anesthetized mouse. (E) Representative traces showing Ca2+ transients (black), bladder pressure (magenta), and EUS-EMG (teal) during fiber photometry recordings, with dashed lines indicating bladder contraction onset. (F) Average Ca2+ signals during bladder contraction from all trials (n = 101 trials from 7 mice). The thick line and shading represent mean ± s.e.m., respectively. (G) Cross-correlation (left) and correlation coefficients (right) between Ca2+ signals and bladder contraction events compared to shuffled data (n = 7 mice, *P = 0.02, Wilcoxon signed-rank test). (H) Cross-correlation (left) and correlation coefficients (right) between Ca2+ signals and EUS-EMG bursting events compared to shuffled data (n = 7 mice, *P = 0.02, Wilcoxon signed-rank test). For all data points in (C, G), and (H), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Inactivation of PMC cells suppresses bladder contraction and EUS bursting activity to suspend voiding.

(A) Schematic of labeling (top), representative histology (middle), and behavior test (bottom) for PMCESR1+ photoinhibition. Scale bar: 100 µm. (B, C) Representative images (B) and quantification (C) of the void area before (‘Pre’), during (‘On’), and after (‘Post’) photoinhibition in PMCESR1-GtACR1 (n = 12 mice) and PMCESR1-mCherry (n = 8 mice) groups (from left to right: ***P = 4.9e-4, ***P = 4.9e-4, P = 0.3, P = 0.5, respectively; n.s., not significant; Wilcoxon signed-rank test). (D) Cumulative trials of voiding duration in PMCESR1-GtACR1 (blue bar, n = 75 trials from 12 mice) and PMCESR1-mCherry (black bar, n = 48 trials from 8 mice) during photoinhibition. Voiding trials are ordered by the increasing time of the voiding epoch with the laser on. (E) Voiding duration before, during, and after photoinhibition in PMCESR1-GtACR1and PMCESR1-mCherry groups (from left to right: ***P = 4.9e-4, ***P = 4.9e-4, P = 0.7, P = 0.9, respectively; n.s., not significant; Wilcoxon signed-rank test). (F) Timeline (top) and schematic (bottom) for PMCESR1+ cells photoinhibition during simultaneous cystometry and electromyography recording. (G) Representative traces (top) and expanded portions (bottom) of bladder pressure (magenta) and EUS-EMG (teal) before, during, and after photoinhibition in PMCESR1-GtACR1 (left) and PMCESR1-mCherry (right) groups. (H, I) Quantification of the Δpressure (H) and EUS-EMG bursting duration (I) during voiding before, during, and after photoinhibition in PMCESR1-GtACR1 (n = 8 mice) and PMCESR1-mCherry (n = 7 mice) groups (H: from left to right: **P = 7.8e-3, **P = 7.8e-3, P = 0.2, P = 0.3, respectively; I: from left to right: **P = 7.8e-3, **P = 7.8e-3, P = 0.8, P = 0.4, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (C, E, H), and (I), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Transection of the pudendal nerves does not impair bladder contraction induced by PMCESR1+ cell photoactivation.

(A) Timeline (top) and schematic (bottom) for PMCESR1-ChR2 photoactivation during simultaneous cystometry and urethral electromyography recordings, with PDNx performed first. PDNx, pudendal nerve transection; PLNx, pelvic nerve transection. (B) Representative traces (top) and expanded portions (bottom, from the dashed box in the top panel) showing bladder pressure (magenta) and EUS-EMG (teal) during PMCESR1-ChR2 photoactivation in various groups, with PDNx performed first. (C) Heatmap (top) and average traces (bottom; thick lines and shading represent mean ± s.e.m.) of sorted bladder pressure and EUS-EMG around the photoactivation timepoint for all unfilled bladder trials with the PDNx-first experiment (n = 8 mice per group). (D, E) Quantification of bladder pressure change (ΔP, D, left), bladder pressure ratio (D, right), the percentage of photoactivation-associated bladder contraction (E, left), and the percentage of photoactivation-associated EUS-EMG bursting (E, right) upon photoactivation for the PDNx-first experiment from (C) (n = 8 mice per group, from left to right, D: P = 0.8, **P = 7.8e-3, P =0.7, **P = 7.8e-3, respectively; E: P = 1, **P = 7.8e-3, **P = 7.8e-3, P = 1, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (D, E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Transection of the pelvic nerves does not impair EUS bursting activity induced by PMCESR1+ cell photoactivation.

(A) Timeline (top) and schematic (bottom) for PMCESR1-ChR2 photoactivation during simultaneous cystometry and urethral electromyography recordings, with PLNx performed first. PLNx, pelvic nerve transection; PDNx, pudendal nerve transection. (B) Representative traces (top) and expanded portions (bottom, from the dashed box in the top panel) showing bladder pressure (magenta) and EUS-EMG (teal) during PMCESR1-ChR2 photoactivation in various groups, with PLNx performed first. (C) Heatmap (top) and average traces (bottom; thick lines and shading represent mean ± s.e.m.) of sorted bladder pressure and EUS-EMG around photoactivation timepoint for all filled bladder trials with the PLNx-first experiment (n = 8 mice per group). (D) Quantification of bladder pressure parameters for the PLNx-first experiment from C: bladder pressure change (ΔP, left), bladder pressure ratio (middle), and the percentage of photoactivation-associated bladder contraction (right; from left to right: **P = 7.8e-3, *P = 0.02, **P = 7.8e-3, P = 0.6, **P = 7.8e-3, P = 1, respectively; n.s., not significant; Wilcoxon signed-rank test). (E) Quantification of EUS-EMG parameters for the PLNx-first experiment from C: EUS-EMG bursting AUC (left), EUS-EMG bursting duration (middle), and the percentage of photoactivation-associated EUS-EMG bursting (right; from left to right: **P = 7.8e-3, **P = 7.8e-3, **P = 7.8e-3, **P = 7.8e-3, P = 1, **P = 7.8e-3, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (D, E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Transection of the pelvic nerves decreases urinary volume induced by PMC cell photoactivation.

(A) Schematic (top) and timeline (bottom) for PMCESR1-ChR2 photoactivation in a freely moving mouse with pelvic nerve transection (PLNx). (B) Representative images of light-induced urination marking (black shading) in ESR1-Cre mice before (‘Baseline’) and after (‘Test’) pelvic nerve transection (left) or sham surgery (right). (C-E) Quantification of the effect of pelvic nerve transection on voiding in the PLNx (n = 9 mice) and sham groups (n = 9 mice): the percentage of light-induced voiding (C, P = 1; n.s., not significant; Wilcoxon signed-rank test), light-induced voiding area (D, **P = 3.9e-3, P = 0.5, respectively), and latency of voiding onset after light stimulation (E, **P = 3.9e-3, P = 0.2, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (D, E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Transection of the pudendal nerves decreases urinary volume induced by PMCESR1+ cell photoactivation.

(A) Schematic (top) and timeline (bottom) for PMCESR1-ChR2 photoactivation in a freely moving mouse with pudendal nerve transection (PDNx). (B) Representative images of light-induced urination marking (black shading) in ESR1-Cre mice before (‘Baseline’) and after (‘Test’) pudendal nerve transection (left) or sham surgery (right). (C-E) Quantification of the effect of pudendal nerve transection on voiding in the PDNx (n = 7 mice) and sham groups (n = 4 mice): the percentage of light-induced voiding (C, P = 1; n.s., not significant; Wilcoxon signed-rank test), light-induced voiding area (D, *P = 1.6e-2, P = 1, respectively), and latency of voiding onset after light stimulation (E, P = 0.6, P = 0.9, respectively; n.s., not significant; Wilcoxon signed-rank test).For all data points in (D, E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Differences in the anatomical projections to the lumbosacral spinal cord between PMCCRH+ and PMCESR1+ cells.

(A) Schematic of labeling (left) and representation histology showing the CTB-647 fluorescence in the lumbosacral spinal cord of CRH-Cre mice. Scale bar: 200 µm. (B, C) Representative image (B) and enlarged images (C, from the left part of B) showing EGFP and mCherry expression in the PMC of a CRH-Cre mouse. Scale bars: 200 µm for (B) and 50 µm for (C). (D) Quantification of the fractions of CRH+ cells specifically projecting to the SPN and DGC of the spinal cord, respectively (n = 2312 cells from 5 mice). (E) Schematics of labeling (left) and representative histology showing the CTB-647 fluorescence in the lumbosacral spinal cord of ESR1-Cre mice. Scale bar: 200 µm. (F, G) Representative image (F) and enlarged images (G, from the left part of F) showing EGFP and mCherry expression in the PMC of an ESR1-Cre mouse. Scale bars: 200 µm for (F) and 50 µm for (G). (H) Quantification of the fractions of ESR1+ cells specifically projecting to the SPN and DGC of the spinal cord, respectively (n = 2468 cells from 7 mice). SPN, sacral parasympathetic nucleus; DGC, dorsal gray commissure.

Coordination of bladder contraction and sphincter relaxation for urination by PMCESR1+ cells.

(A) Left: Example (left, with arrows indicating the onset timepoints) and quantification (right) of the temporal relationships among the onset times of Ca2+ signals, bladder pressure (BP) upstroke, and EUS-EMG bursting in photometry recordings (n = 46 trials from 8 mice; Ca2+signals onset, 0\0-0 s; BP upstroke onset, 0.6\0.3-1.2 s; EUS-EMG bursting onset, 2.3\1.5-3.2 s; ***P = 3.5e-9 for all, Wilcoxon signed-rank test). (B) Example (left, with arrows indicating the onset timepoints) and quantification (right) of the temporal relationships among the onset times of light stimulation, bladder pressure upstroke, and EUS-EMG bursting for the photoactivation group (n = 50 trials from 10 mice; Light on, 0\0-0 s; BP upstroke onset, 0.4\0.4-0.5 s; EUS-EMG bursting onset, 0.8\0.7-1.0 s; ***P = 7.6e-10 for all, Wilcoxon signed-rank test). (C) Example (left, with arrows indicating the onset timepoints) and quantification (right) of the temporal relationships among the onset time of light, EUS-EMG bursting end, and bladder pressure upstroke end for the photoinhibition group (n = 44 trials from 8 mice; Light on, 0\0-0 s; EUS-EMG bursting end, 0.1\0.1-0.2 s; BP upstroke end, 0.8\0.8-0.9 s; ***P = 7.6e-9 for all, Wilcoxon signed-rank test). (D) A working model of the brainstem-spinal compound circuit for bidirectional and coordinated control of voiding. Abbreviations: SPN, sacral parasympathetic nucleus; DGC, dorsal gray commissure; DL, dorsolateral nucleus; MPG, major pelvic ganglia. The guarding reflex is triggered by bladder afferents via the pelvic nerves and mediated by spinal interneuronal circuits, which activate the urethral sphincter to prevent involuntary bladder emptying (Fowler et al., 2008). For all data points in (A, B), and (C), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

The activity of PMC neurons increases during successful voiding, related to Figure 1.

(A) Schematic (left) of labeling and representative histology (middle) of PMCESR1+ cells labeled with GCaMP6f. Scale bar: 100 µm. TH (tyrosine hydroxylase) stained neurons in the locus coeruleus. Right: Overlay of GCaMP6f-labelled areas from 9 mice. Scale bar: 200 µm. (B) Schematic of fiber photometry Ca2+ recording in a freely moving mouse. (C) Representative Ca2+ traces (top) and voiding events (bottom, yellow circle). (D) Cumulative sessions of Ca2+ signals aligned to voiding onset (dotted line). (E) Boxplots showing the amplitude of voiding-related Ca2+ signals in various groups (n = 9 mice per group, 31.8%\26.7%-33.2% for Gcamp6f, 5.4%\3.7%-6.0% for Shuffled, 4.7%\2.5%-5.7% for EYFP, **P = 3.9e-3 (Gcamp6f versus Shuffled), Wilcoxon signed-rank test; ***P = 4.1e-5 (Gcamp6f versus EYFP), Wilcoxon rank-sum test). (F) Detected voiding-related Ca2+ events in various groups (n = 9 mice per group, 100%\100%-100% for Gcamp6f, 0%\0%-0% for Shuffled, 0%\0%-0% for EYFP. **P = 3.9e-3 (Gcamp6f versus Shuffled), Wilcoxon signed-rank test; ***P = 4.1e-5 (Gcamp6f versus EYFP), Wilcoxon rank-sum test). For all data points in (E, F), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

No change in fluorescence responses of EYFP-labeled PMC cells during voiding.

(A) Schematics of labeling (left) and representative histology (right) of PMCESR1+ cells labeled with EYFP. Scale bar, 100 µm. (B) Cumulative sessions of fluorescence responses aligned to voiding onset (n = 248 trials from 9 mice). (C) Average fluorescence traces of PMCESR1-EYFP aligned to voiding onset. The black line and shading represent mean ± s.e.m., respectively.

Identification of recorded PMCESR1+ cells in optrode recordings.

(A) Tetrode locations for all recorded PMCESR1+ units (red dots, n = 28 units from 4 mice). (B) Left: Raster plots (top) and histograms (bottom) showing the firing patterns of representative PMCESR1-ChR2 units upon optical stimulation at a power of 10 mW. Right: Waveforms of light-induced (red) and spontaneous (gray) spikes from the unit shown on the left. (C) Distribution of correlation coefficients between spontaneous and light-induced spikes across all recorded PMCESR1+ units. (D) Comparison of success rates and temporal jitter for the first light-induced spike in all recorded PMCESR1+ units. (E) Latency distribution for all recorded PMCESR1+ units. (F, G) Representative firing patterns of opto-tagged PMCESR1+ cells. F: Increased activity in all trials (left, middle) and in a subset of trials (right) during urination; G: No increase in activity during urination. Top: Raster plots. Bottom: Average firing rates.

No voiding contractions (NVCs) correlate with Ca signals of PMC cells.

(A) Representative traces of Ca2+ transients (black), bladder pressure (magenta), and EUS-EMG (teal) for no voiding contractions (NVCs) and voiding contractions (VCs). (B) Left: Representative fiber-photometry trace aligned to an EUS bursting episode. Right: Expanded view of the boxed region showing a small Ca²⁺ signal hump before bursting onset (yellow arrow) and a continuous rise during the bursting. (C) The percentage of NVCs (n = 62 events from 3 mice) and VCs events (n = 167 events from 3 mice). (D) Correlation rate of Ca2+ transient with bladder contraction and EUS-EMG bursting events (n = 3 mice per group). (E) Comparison of peak bladder pressure between NVCs and VCs (NVCs: n = 62 events from 3 mice, VCs: n = 167 events from 3 mice; ***P = 6.03e-7, Wilcoxon rank-sum test). (F) Quantification of peak Ca²⁺ signals during NVCs and VCs, and Ca²⁺ signal amplitudes at bursting onset and offset during VCs (NVCs: 62 events from 3 mice, VCs: 79 events from 3 mice; ***P = 8.2e-7, NVC peak versus VC peak, and ***P = 4.4e-4, NVC peak versus VC bursting offset, Wilcoxon rank-sum test; ***P = 1.1e-14, VC bursting onset versus VC bursting offset, Wilcoxon signed-rank test). For all data points in (E, F), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Acute photoinhibition (60 s) of PMC cells operates the bladder and sphincter to suspend voiding, related to Figure 2.

(A) Overlay of viral expression areas (top) and fiber positions (bottom) from ESR1-Cre mice labeled with GtACR1 (n = 12 mice). (B) Latency of voiding suspension after light activation. (C) Representative raw traces of bladder pressure and EUS-EMG. (D) Latency of sphincter bursting termination after light activation (n = 8 mice). (E) Bladder threshold pressure during voiding before (‘Pre’), during (‘On’), and after (‘Post’) photoinhibition in PMCESR1-GtACR1 (n = 8 mice) and PMCESR1-mCherry (n = 7 mice) groups (from left to right: P = 0.06, **P = 7.8e-3, P = 0.9, P = 0.4, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (B, D) and (E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

5-s photoinhibition of PMC cells suppresses bladder contraction and EUS bursting activity to suspend ongoing voiding.

(A) Experimental design for 5 s photoinhibition of PMCESR1+ cells in a freely moving mouse. (B, C) Representative images (B, blue and black shading) and quantification (C) of the void area before, during, and after 5 s photoinhibition in PMCESR1-GtACR1 (n = 12 mice) and PMCESR1-mCherry (n = 8 mice) groups (from left to right: ***P = 4.9e-4, ***P = 4.9e-4, P = 0.7, P = 0.9, respectively; n.s., not significant; Wilcoxon signed-rank test). (D) Cumulative trials of voiding duration during 5 s photoinhibition in PMCESR1-GtACR1 (blue bar, n = 65 trials from 12 mice) and PMCESR1-mCherry groups (black bar, n = 38 trials from 8 mice), ordered by increasing voiding epoch time with the laser on. (E) Voiding duration before, during, and after 5 s photoinhibition in PMCESR1-GtACR1 (n = 12 mice) and PMCESR1-mCherry (n = 8 mice) groups (from left to right: ***P = 4.9e-4, ***P = 4.9e-4, P = 0.3, P = 0.4, respectively; n.s., not significant; Wilcoxon signed-rank test). (F) Latency of urination suspension after light activation. (G) Representative traces (left) and expanded portions (right, from the dashed box in the left panel) of bladder pressure (magenta) and EUS-EMG (teal) before, during, and after 5 s photoinhibition in PMCESR1-GtACR1 individual. (H-L) Quantification of the effect of 5 s photoinhibition (n = 33 trials from 6 mice) on bladder pressure and EUS-EMG: Δpressure (H, ***P = 5.4e-7), threshold pressure (I, P = 0.98, ***P = 1.9e-6, respectively), EUS-EMG bursting AUC (J, ***P = 5.4e-7), and EUS-EMG bursting duration (K, ***P = 5.4e-7; n.s., not significant; Wilcoxon signed-rank test). (L) Latency of sphincter bursting termination after 5 s photoinhibition. For all data points in (C), (E), (F), and (H-L), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Activation of PMC cells induces both bladder contraction and EUS bursting activity to initiate voiding.

(A) Schematics of labeling (left), representative histology (middle), and behavior test (right) for PMCESR1+ photoactivation. Scale bar: 200 µm. (B) Cumulative trials of voiding duration in PMCESR1-ChR2 (blue bar, n = 172 trials from 8 mice) and PMCESR1-mCherry (black bar, n = 166 trials from 8 mice) photoactivation. Voiding trials are ordered by the latency of the voiding epoch with the laser on. (C) % of photoactivation-associated voiding events in PMCESR1-ChR2 and PMCESR1-mCherry groups (n = 8 mice per group, ***P = 1.6e-4, Wilcoxon rank-sum test). (D) Latency of voiding onset after light on. (E) Timeline (top) and schematics (bottom) for PMCESR1+ cells photoactivation during simultaneous cystometry and electromyography recording. (F) Representative traces (top) and expanded portions (bottom) of bladder pressure (magenta) and EUS-EMG (teal) around photoactivation timepoint in PMCESR1-ChR2(left) or PMCESR1-mCherry (right) groups. (G, H) Quantification of bladder pressure change (ΔP, G, left), the ratio of bladder pressure (G, right), the percentage of photoactivation-associated bladder contraction (H, left), and the percentage of photoactivation-associated EUS-EMG bursting (H, right) upon photoactivation (n = 9 PMCESR1-ChR2 mice, n = 6 PMCESR1-mCherry mice, ***P = 4e-4 for (G, H), Wilcoxon rank-sum test). For all data points in (C, D, G), and (H), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Regular interval photoactivation of PMC cells induces both bladder contraction and EUS bursting activity.

(A) Timeline (top) and schematic (bottom) for regular interval photoactivation of PMCESR1+ cells during simultaneous cystometry and urethral electromyography recording with a filled bladder. (B) Representative raw traces of bladder pressure (magenta) and EUS-EMG (teal) around the photoactivation timepoint in PMCESR1-ChR2 (left) and PMCESR1-mCherry (right) individuals. (C) Average bladder pressure around photoactivation in PMCESR1-ChR2 (left, n = 8 mice) and PMCESR1-mCherry (right, n = 8 mice) groups. The thick line and shading represent mean ± s.e.m., respectively. (D, E) Quantification of the photoactivation effect on the bladder detrusor and urethral sphincter in PMCESR1-ChR2 (n = 8 mice) or PMCESR1-mCherry (n = 8 mice) groups: the percentage of bladder contraction (D, left), the percentage of EUS-EMG bursting (D, right), Δpressure (E, left), and bladder pressure ratio (E, right; ***P = 1.6e-4 for D and E, Wilcoxon rank-sum test). For all data points in (D, E), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Transection of the pelvic nerves does not alter bladder pressure in an unfilled bladder during PMCESR1-ChR2 photoactivation.

(A) Timeline (top) and schematic (bottom) for PMCESR1-ChR2 photoactivation during simultaneous cystometry and urethral electromyography recordings in a non-filled bladder, with PLNX performed first. PLNx: pelvic nerve transection; PDNx: pudendal nerve transection. (B) Representative traces (top) and expanded portions (bottom, from the dashed box in the top panel) showing bladder pressure (magenta) and EUS-EMG (teal) during PMCESR1-ChR2 photoactivation in an unfilled bladder with PLNX performed first. (C) Heatmap (top) and average traces (bottom, thick line, and shading represent mean ± s.e.m., respectively) of sorted bladder pressure and EUS-EMG around photoactivation timepoint for all unfilled bladder trials with PLNX performed first (n = 8 mice per group). (D, E) Quantification of bladder pressure change (ΔP, D, left), bladder pressure ratio (D, right), the percentage of photoactivation-associated EUS-EMG bursting (E, left), and the percentage of photoactivation-associated bladder contraction (E, right) upon photoactivation for the PLNx-first experiment from C (n = 8 mice per group, from left to right, D: **P = 7.8e-3, P =0.8, **P = 7.8e-3, P = 0.1, respectively; E: P = 1, **P = 7.8e-3, **P = 7.8e-3, P = 1, respectively; n.s., not significant; Wilcoxon signed-rank test). For all data points in (D, E) whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

PMC cells projection to the SPN and DGC in the spinal cord.

(A) Left: Schematic of labeling. Middle and right: Representative histological images showing CTB-555 expression in the lumbosacral spinal cord (middle) and mGFP expression in the PMC (right). Scale bars: 200 µm. (B) Axonal projections of PMCESR1-mGFP cells in the lumbosacral spinal cord from L5 to S2 levels (n = 3 mice). Scale bars: 200 µm. Abbreviations: SPN, sacral parasympathetic nucleus; DGC, dorsal gray commissure.

Dual-projecting PMC neurons are active throughout voiding, correlating with bladder pressure and EUS bursting, and their optogenetic activation initiates urination.

(A) Left: Schematic of the labeling strategy. Right: Representative histological images showing GCaMP6s expression in the PMC and CTB-555 labeling in the lumbosacral spinal cord of a wild-type mouse. Scale bars: 100 µm (top), 200 µm (bottom). (B) Cumulative sessions of Ca2+ signals aligned to voiding onset (dotted line). (C) Quantification of voiding-related Ca2+ events from 4 mice. (D) Boxplots showing the amplitude of voiding-related Ca2+ signals in various groups (n = 102 trials from 4 mice per group, 5.0%\3.8%-6.0% for GCaMP6s, 0.6%\0.4%-0.8% for shuffled, ***P = 1.8e-18, Wilcoxon signed-rank test). (E) Schematic of fiber photometry recording for dual-projecting PMC cells during simultaneous cystometry and EUS-EMG in a urethane-anesthetized mouse. (F) Representative traces showing Ca2+ transients (black), bladder pressure (magenta), and EUS-EMG (teal) during fiber photometry recordings. Dashed lines indicate the onset of bladder contractions. (G) Cross-correlation (left) and correlation coefficients (right) between Ca2+ signals and bladder contraction events, compared to shuffled data (n = 40 trials from 4 mice, ***P = 3.6e-8, Wilcoxon signed-rank test). (H) Cross-correlation (left) and correlation coefficients (right) between Ca2+ signals and EUS-EMG bursting events, compared to shuffled data (n = 40 trials from 4 mice, ***P = 3.6e-8, Wilcoxon signed-rank test). (I) Left: Schematic of the labeling strategy. Right: Representative histological images showing hChR2 expression in the PMC and CTB-488 labeling in the lumbosacral spinal cord of a wild-type mouse. Scale bars: 100 µm (top), 200 µm (bottom). (J) Cumulative trials of voiding duration in dual-projecting PMC cells photoactivation (blue bar, n = 94 trials from 4 mice). Voiding trials are ordered by the latency of the voiding epoch with the laser on. (K) Latency of voiding onset after light on. (L) Schematic for dual-projecting PMC cells photoactivation during simultaneous cystometry and EUS-EMG in a freely behaving mouse. (M) Representative traces and expanded portion (black dashed box) of bladder pressure (magenta) and EUS-EMG (teal) around photoactivation timepoint. Dashed line indicates bladder contraction onset. (N) The percentage of photoactivation-associated bladder contraction and EUS-EMG bursting activity (n = 3 mice per group). For all data points in (C, D, G, H, K), and (N), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.

Dual-projecting PMC neurons coordinate bladder contraction and EUS bursting activity.

(A) Left: Example (left, with arrows indicating the onset timepoints) and quantification (right) of the temporal relationships among the onset times of Ca2+ signals, bladder pressure (BP) upstroke, and EUS-EMG bursting in photometry recordings (n = 40 trials from 4 mice; Ca2+signals onset, 0\0-0 s; BP upstroke onset, 1.2\0.9-1.6 s; EUS-EMG bursting onset, 2.3\2.0-3.3 s; ***P = 3.6e-8 for all, Wilcoxon signed-rank test). (B) Example (left, with arrows indicating the onset timepoints) and quantification (right) of the temporal relationships among the onset times of light stimulation, bladder pressure upstroke, and EUS-EMG bursting for the photoactivation group (n = 30 trials from 3 mice; Light on, 0\0-0 s; BP upstroke onset, 0.7\0.5-0.8 s; EUS-EMG bursting onset, 1.0\0.9-1.2 s; ***P = 1.7e-6 for all, Wilcoxon signed-rank test). For all data points in (A, B), whisker-box plots indicate the median with the 25%-75% percentile as the box, and whiskers represent the minimum and maximum values.