Central Control Circuit for Context-dependent Micturition

Context and Social Hierarchy Dependent Micturition Behavior in Male Mice

Male mice modulate their micturition patterns depending on factors such as social rank (Desjardins et al., 1973) and olfactory cues (Kaur et al., 2014), indicating the existence of neural circuitry relaying higher order brain function to bladder control. We examined if similar environmental and experiential control of urine release occurs in C57BL/6N mice in a laboratory setting. Pair-housed adult C57BL/6N male mice were separated into individual cages lined with filter paper with an olfactory stimulus (estrous female urine or saline) added to the cage center and rubbed onto the nose (Fig. 1A). After 2 hr in the cage, mice were removed, tested for social hierarchy with their previous cage mate using the tube test (Lindzey et al., 1961), and the brains were processed for histology. In parallel, the distribution of urine spots on the paper was examined using fluorescence imaging.

Among the four categories (2-way combinations of dominant/subordinate and female urine/saline), only mice that are both dominant and exposed to female urine scattered a large number of urine spots throughout the arena (Fig. 1A). In response to female urine, dominant mice deposit significantly more spots than their subordinate cage mates (dominant: 119±19 spots, subordinate: 36±24 spots, mean±SEM; n=6 mice each, p=0.024) (Fig. 1B). This effect was not seen in response to saline (dominant: 31±12 spots, subordinate 23±6 spots, n=4 mice each, p>0.5). In addition, dominant mice exposed to female urine deposit urine spots throughout the cage and preferentially toward the female urine center whereas in the other contexts mice deposit urine further from the stimulus center (average urine spot distance to stimulus center, dominant with female urine: 8.1±0.5 cm, subordinate with female urine: 11.2±0.7 cm, dominant with saline: 11.6±0.4 cm, subordinate with saline: 12.1±0.8 cm; p=0.017, Kruskal-Wallis test) (Fig. 1C). Furthermore, real-time tracking of mouse position (Fig. S1AB) and micturition position (Fig. S1CD) in an open-field arena indicates that the average location of the urine spots is not predicted by the average location of the mouse.

These findings suggest the existence of a neural circuit in the mouse brain that integrates multiple factors, include social rank and sensory cues, to regulate micturition. A candidate region is the brainstem nucleus PMC. In order to determine if the activity of PMC correlates with degrees of marking behavior, we used the protein expression of immediate early gene c-fos as a read-out of neural activation after marking (Fig. 1D). The PMC was identified based on immunolabeling of the neighboring locus coeruleus (LC) for tyrosine hydroxylase (TH). The numbers of c-fos+ cells in PMC and of urine spots deposited per animal positively correlate (n=20 mice, R²=0.56), suggesting a link between PMC activity and micturition behavior. The numbers of c-fos+ cells are also higher in LC of mice exposed to female urine (e.g. Fig. 1D) but this was not analyzed further here.

Neuronal Subpopulations within the PMC

The PMC contains neurons that express Crh and are labeled by retrograde tracers injected into the sacral spinal cord (Keegan et al., 1994; Valentino et al., 1996; Vincent and Satoh, 1984). We were able to visualize the PMC in coronal brainstem slices of transgenic mice produced by crossing Crh^ires-Cre (Taniguchi et al., 2011) and Cre-dependent tdTomato reporter (Rosa26^lsl-tdTomato) mice. In these slices, a collection of Cre-expressing (Cre+) cells marked by tdTomato is located medial to and juxtaposed with the LC, consistent with the described location of the PMC (Franklin and Paxinos, 2007) (Fig. 2A). In situ hybridization confirmed Cre expression in PMC Crh+ neurons in the Crh^ires-Cre mice: 99% of Crh+ cells express Cre, and 90% of Cre+ cells express Crh (n=179 cells, 2 mice, Fig. S2A). Furthermore, staining with general (DAPI) and neuron-specific (NeuN) nuclear markers revealed that each PMC contains a total of ~1900 cells and ~1100 neurons, with ~44% of neurons being Crh+ (n=2 mice, Fig. 2B).

To determine if the Crh+ neurons, the subset of PMC cells that can be uniquely identified and manipulated using the Crh^ires-Cre transgenic mouse line, are functionally distinct from their Crh− neighbors, we characterized the intrinsic electrophysiological profiles of PMC neurons with whole-cell current-clamp recordings in acute coronal brainstem slices from P17-P21 Crh^ires-Cre::Rosa26^lsl-tdTomato mice (Fig. S2C). Compared to Crh− neurons, Crh+ neurons have lower R_m (Crh+: 463±66, n=10 cells; Crh−: 734±80 MΩ, n=8 cells; p=0.027), higher C_m (Crh+: 108±9; Crh−: 48±7 pF; p=0.0003), lower induced firing rates (Crh+: 7.8±1.1; Crh−: 26.4±4.7 Hz with 100 pA current injection; p=0.0015), and lower voltage sag amplitude in response to −100 pA current injection (Crh+: 1.8±1.3; Crh−: 8.0±2.3 mV; p=0.020) (Fig. S2DE). Principal component (PC) analysis of the electrophysiological parameters permitted good separation between Crh+ and Crh− cells (Fig. S2F; first PC explained 86% of the variance). Thus, PMC Crh+ and Crh− neurons have distinct passive and active cellular properties, with further heterogeneity within each group.

Crh+ PMC Neurons are Glutamatergic and Project to the Sacral Spinal Cord

To determine if Crh+ neurons constitute a bladder-controlling output of the brainstem, we investigated the neurotransmitter identity and anatomy of these neurons. In situ hybridization was performed to detect mRNA of Crh, vesicular glutamate transporters (Vglut, encoded by Slc17a7, Slc17a6, and Slc17a8), and glutamic acid decarboxylases (Gad, encoded by Gad1 and Gad2) (Fig. 2C). Single cell mRNA counts of Crh correlated with those of Vglut, and anti-correlated with those of Gad (Fig. 2D). Nearly all Crh+ cells are glutamatergic (Vglut+ 94%, n=444 cells, 2 mice, Fig. 2E), whereas the Crh− cells are a mix of glutamatergic (Vglut+ 53%) and GABAergic (GAD+ 45.2%). In situ hybridization using tissue from transgenic mice that express fluorophores in glutamatergic or GABAergic neurons yielded similar results (Fig. S2B). The Crh mRNA count in each cell serves as a good classifier to differentiate between glutamatergic and GABAergic cells in the PMC such that high Crh counts indicate that a cell is glutamatergic. Using receiver-operating characteristic (ROC) analysis, in which cells with Crh mRNA counts above a certain threshold are classified as glutamatergic, results in area under curve (AUC) of 0.84 (Fig. 2D), closer to a perfect classifier (AUC=1) than a “coin-toss” classifier (AUC=0.5). Thus, Crh expression is strongly correlated with and predictive of expression of glutamatergic markers in the PMC.

To determine if Crh+ neurons of the PMC project to the spinal cord, we exploited human placental alkaline phosphatase (PLAP), a glycophosphatidylinositol anchored enzyme that robustly transports along neuronal processes and whose enzymatic activity is easily detected. A plasmid encoding a double-floxed inverted open reading frame (DIO) of PLAP under transcriptional control of the CAG promoter (AAV-DIO-PLAP; Fig. 2F) was generated to produce recombinant adeno-associated virus (AAV) that expresses PLAP in a Cre-dependent manner. AAV-DIO-PLAP injected unilaterally into the PMC of Crh^ires-Cre mice yielded labeling of axon terminals bilaterally at L6/S1 level in the spinal cord, the location of the sacral parasympathetic nucleus (n=4 mice) (Fig. 2F). Thus, Crh+ cells comprise half of the PMC neuronal population, are glutamatergic, and project to the sacral spinal cord.

Activating PMC Crh+ Neurons Triggers Bladder Contractions

The finding that glutamatergic Crh+ neurons in PMC send long-range projections to spinal cord supports the hypothesis that Crh+ PMC neurons are output neurons from the brain that control the bladder. If this is the case, then activating these neurons should be sufficient to evoke bladder contraction, and the activity of these neurons should correlate with intrinsic bladder activity. Furthermore, silencing these neurons should disrupt micturition behavior.

To address the first prediction, we investigated the effect of activating Crh+ PMC neurons on bladder function. We expressed the light-activated cation channel channelrhodopsin-2 (ChR2) selectively in Crh+ PMC neurons using Cre-dependent AAV (AAV-DIO-ChR2-tdTomato) and monitored bladder contractions using a catheter surgically inserted into the bladder (Fig. 3A). Saline infusion into the bladder of an anesthetized mouse slowly fills the bladder at a constant rate, which triggers first non-micturating contractions followed by a micturating contraction. The latter is detected as a spike in bladder pressure followed by a fall in pressure to the lowest point reflecting the now empty bladder (Uvin et al., 2012). Although the pressure spikes vary from mouse to mouse, the general pattern described above is preserved across mice. We recorded bladder pressure while light (473 nm, 15 ms pulses x 20 Hz for 5 s, delivered every 60 s) was delivered via an optic fiber implanted unilaterally above the PMC. In control mice expressing only tdTomato in Crh+ PMC neurons (AAV-DIO-tdTomato), light delivery did not trigger bladder contraction (Fig. 3A). In contrast, in mice expressing ChR2-tdTomato in PMC Crh+ neurons, light delivery triggered time-locked bladder contraction in a majority of the trials, on top of the intrinsic bladder rhythm (Fig. 3B). In separate trials, light was delivered at randomized intervals between 30 and 90 s to prevent possible entrainment of bladder contracture to the fixed 60 s interval used above. Light triggered time-locked contractions were again seen in a majority of trials (Fig. S3).

Alignment of bladder pressure recordings to the onset of light delivery revealed increases in pressure occurring during and outlasting light delivery in ChR2-expressing animals, but not in control tdTomato-expressing mice (Fig. 3C). Bladder pressure was higher at the end of the 5 s light stimulus (P_5sec) compared to before the stimulus (P_pre), as seen across all trials in an example mouse (Fig. 3D) and on average for 5 of 6 mice analyzed (ChR2: P_5sec/P_pre=1.30±0.11, n=6 mice; tdTomato control: 0.96±0.02, n=4 mice; p=0.041). Thus, activation of PMC neurons is sufficient to trigger time-locked bladder contraction.

Activity of PMC Crh+ Neurons Tracks Bladder Contraction and Micturition

To address the second prediction that the activity of Crh+ PMC neurons is correlated to bladder contraction, we simultaneously monitored PMC activity and bladder pressure in anesthetized mice. Genetically encoded Ca²⁺ indicator GCaMP6s (Chen et al., 2013) was expressed in Crh+ PMC neurons use Cre-dependent viruses (AAV-DIO-GCaMP6s). Fiber photometry (Cui et al., 2013; Gunaydin et al., 2014) was subsequently used to monitor activity-dependent Ca²⁺ entry in Crh+ PMC neurons in vivo. A fiber optic was implanted in PMC and used to deliver 473 nm excitation light and collect fluorescence emission. Bladder contractions were measured with cystometry as described above (Fig. 4A). As predicted, simultaneous recordings revealed that GCaMP6s fluorescence intensity, reflecting Ca²⁺ influx into Crh+ PMC neurons, correlated with spike-like increases in bladder pressure, reflecting bladder contractions (Fig. 4B). Aligning GCaMP6s fluorescence to the onset of bladder contraction (time=0 defined as pressure reaching 5 cmH₂O above baseline) showed that Ca²⁺ transients in PMC Crh+ neurons were time-locked to bladder contraction (Fig. 4C). Furthermore, cross-correlation of fluorescence and bladder pressure revealed a mean peak correlation coefficient higher than the shuffled data (data: 0.62±0.07, shuffled: −0.01±0.02, mean±SEM, n=4 mice, p=0.0286) (Fig. 4D).

To determine if micturition in freely moving mice coincides with activation of Crh+ PMC neurons, we simultaneously monitored mouse locomotion, urine deposition, and Ca²⁺ entry into Crh+ PMC neurons. Urine deposition was visualized by fluorescence with a blue excitation LED and a video camera (with GFP filter set) under the arena, whereas mouse position and posture were captured with an infrared (IR) LED and video camera above the arena (Fig. 4E). To encourage micturition in this foreign and brightly lit arena, mice were injected with the diuretic furosemide. GCaMP6s fluorescence was collected with fiber photometry as described above while the mice moved freely, revealing that fluorescence increases at times of urine deposition (Fig. 4F). On average and in individual micturition events, GCaMP6s fluorescence from Crh+ PMC neurons rapidly increased before micturition, an effect absent in shuffled data (data: max ΔF/F=4.9; shuffled: 1.3; 75 micturition events, n=4 mice) (Fig. 4G, 4H). Furthermore, 2-color fiber photometry with the addition of a reference red channel in Crh^ires-Cre::Rosa26^lsl-tdTomato animals injected with Cre-dependent GCamp6s showed that intensity of GCamp6s fluorescence, but not tdTomato, positively correlated with micturition (Fig. S4), indicating that the correlated GCamp6s signal could not be explained by movement artifacts. Thus, the bulk activity of Crh+ PMC neurons correlates with bladder pressure under anesthesia and is synchronized with initiation of micturition in freely moving mice.

Reducing Activity of Crh+ PMC Neurons Impairs Micturition Behavior

To test the requirement of activity in Crh+ PMC neurons for micturition, we used the chemogenetic tool hM4D_i, an engineered G_i protein-coupled receptor activated by the inert ligand clozapine-N-oxide (CNO), to reduce the activity of Crh+ PMC neurons while adult male mice were exposed to female urine as in Fig. 1 (AAV-hM4Di-mCherry, Fig. 5A). Analysis of consecutive trials with IP injection of either CNO (2 trials) or saline (2 trials) in randomized order demonstrated disrupted micturition in CNO trials (Fig. 5B). Both the number of urine marks and the total area marked on filter paper, indicative of the total micturition volume, significantly decreased in CNO trials compared to saline trials (number of marks: p=0.0024; total area marked: p=0.027; n=12, Fig. 5C). In contrast CNO injection did not affect micturition in wildtype mice (number of marks: p=0.38; total area marked: p=0.38; n=12, Fig. S5B). Furthermore, despite the decrease in total urine output in CNO-treated Crh^ires-Cre mice, the spatial distribution of the remaining urine marks, as indicated by their average distance to stimulus center, was unaffected (p=0.37; n=11 mice; one mouse with zero urine marks in CNO was excluded from the analysis of urine mark distribution, Fig. S5B). These results demonstrate that activation of hM4D in Crh+ PMC neurons reduces the amount but not the pattern of urine release in freely-moving mice, consistent with the hypothesis that activity of these cells normally drives micturition in the marking assay.

Crh+ PMC Neurons Receive Converging Inputs from the Brain

The results presented above indicate that Crh+ neurons are descending command neurons that control bladder function. Thus, they are poised, anatomically and functionally, to integrate pro-micturition and anti-micturition inputs from relevant brain areas, and transduce these changes into urine output. To identify candidate neurons throughout the brain that synapse onto Crh+ PMC neurons, we used rabies-based retrograde trans-synaptic labeling (Wall et al., 2010; Wickersham et al., 2007). The number and distribution of candidate neurons presynaptic to Crh+ PMC neurons were determined with an unbiased and automated method that uses serial 2-photon tomography (STP) and 3D reconstruction to quantitatively map rabies-labeled neurons (Ragan et al., 2012) (Fig. 6A).

As control experiments for the specificity of rabies infection to Crh+ neurons and candidate inputs, injection of glycoprotein-deleted, EGFP-encoding rabies virus pseudotyped with EnvA (RbV-EGFP) in Crh^ires-Cre mice without helper viruses (AAV-DIO-TVA-mCherry and AAV-DIO-RG) showed no rabies infection in the injection site (Fig. S6A), whereas omitting AAV-DIO-RG resulted in robust starter cell labeling in the PMC but no EGFP+ neurons elsewhere in the brain (Fig. S6B). PMC and nearby areas were separately examined for the specificity of the starter cells (Fig. 6B). Furthermore, as a control experiment to differentiate inputs to PMC from those to medial vestibular nucleus (MV), a nucleus containing Crh+ neurons posterior to the PMC and hence a potential source of starter cell contamination, Crh+ MV neurons were specifically targeted as starter cells. This experiment, using both helper viruses AAV-DIO-TVA-mCherry and AAV-DIO-RG as well as RbV-EGFP, revealed no significant overlap between the EGFP-labeled candidate inputs to MV (Fig. S6C) and those to PMC described below.

Among 14 mice with robust and specific RbV starter cell labeling, 11 brains (6 males and 5 females) were sliced and examined for RbV labeled areas, and 3 brains (2 males and 1 female) were processed for whole-brain STP cell counting. In all 14 mice, dense candidate input cells to PMC Crh+ cells were observed in prefrontal areas including anterior cingulate cortex (ACA) and prelimbic cortex (PL), as well as motor cortex (MO) and somatosensory cortex (SS). More posterior labeled areas included the bed nucleus of the stria terminalis (BST), including both anterior and posterior divisions (BSTa and BSTp), medial preoptic area (MPOA), paraventricular hypothalamic nucleus (PVH), lateral hypothalamus (LHA), posterior hypothalamus (PH), and zona incerta (ZI). Central amygdalar nucleus (CEA) also contained dense labeling, as did midbrain regions including periaqueductal gray (PAG), superior colliculus motor-related (SCm) and midbrain reticular nucleus (MRN) (Fig. 6C). These areas are a subset of the 27 brain regions identified with whole-brain STP cell counting that each contain at least 0.1% of total EGFP+ cells within each mouse (Table S1). Although the numbers of EGFP+ cells throughout the brain varied across mice (3731–39355 cells; n=3 mice), the fractions of cells labeled per region within each macro structure (cortex, cerebral nuclei, hypothalamus, midbrain) were strikingly consistent (Fig. 6D).

Inhibiting MPOA GABAergic Neurons Modifies Micturition Patterns and Prevents Rank-dependent Differences

A robust putative input to Crh+ PMC neurons implicated by rabies tracing experiments is MPOA, a heterogeneous structure connected to multiple regions implicated in social behaviors (Simerly and Swanson, 1986, 1988; Simerly et al., 1986). In situ hybridization revealed that the rabies-labeled neurons in MPOA are largely Gad+ (including both Gad1 and Gad2, 89.9%, n=198 cells/3 mice, Fig. S7AB), suggesting that MPOA mainly sends GABAergic input to Crh+ PMC neurons. To test the hypothesis that GABAergic MPOA neurons regulate micturition in a marking context, we inhibited bilateral MPOA neurons expressing the vesicular GABA transporter (VGAT, encoded by Slc32a1) by injecting AAV-DIO-hM4Di-mCherry into MPOA of Slc32a1^ires-Cre males, and tested their micturition pattern in response to female urine (Fig. 7A). With CNO injection, the size of urine marks increased for both dominant and subordinate males (Fig. 7B). Inhibiting MPOA VGAT+ neurons significantly decreased the number of urine marks while increasing the average size of each mark, leaving the total area marked unchanged (number of marks: p=0.011; total area marked: p=0.64; average mark size: p=0.0012; n=13 mice, Fig. 7C, Fig. S7C). Furthermore, inhibiting VGAT+ MPOA neurons negates the differences in micturition behavior between dominant and subordinate males that were present in saline trials (number of marks: saline, p=0.0012; CNO, p=0.38; average mark size: saline, p=0.0082; CNO, p>0.99. n=13 mice; Fig. 7D). These results are consistent with a model in which silencing inhibitory MPOA inputs dis-inhibits Crh+ PMC neurons, and that GABAergic MPOA neurons normally modulate micturition in the marking assay. Thus, these neurons influence both social-rank dependent micturition patterns and the amount of urine released per bladder contraction.

Cellular Composition of the PMC

Single-unit recordings in dorsolateral pons in cats and rats demonstrated the existence of “direct neurons” whose firing rate increases as bladder contracts (de Groat et al., 1998; Sasaki, 2004, 2005). Intermingled with these neurons were “inverse neurons” that could not be stimulated antidromically from spinal cord and whose firing rate decreases as bladder contracts. We find that, in mice, population activity of Crh+ PMC neurons increases with bladder contraction, suggesting that these anatomically and molecularly defined neurons likely correspond to the functionally defined “direct neurons”. Furthermore, we find that in awake and freely moving mice, PMC Crh+ neurons increase activity during urine release, indicating a correlation of their activity not just with bladder contraction but also with micturition, which requires contraction of the bladder wall as well as relaxation of the urinary sphincters. Although not tested here, we hypothesize that “inverse neurons” could correspond to GABAergic Crh− neurons of the PMC which may represent inhibitory interneurons.

The elevated activity of direct neurons and of PMC Crh+ neurons during bladder contractions could be caused by ascending sensory signals conveying bladder pressure to the brain stem or could reflect the command signals that trigger bladder contractions. Electrical stimulation in the vicinity of the PMC is sufficient to trigger bladder contractions in cats (Noto et al., 1989), suggesting the existence of pro-micturition neurons in the pons. We found that ChR2-mediated activation of PMC Crh+ neurons triggered time-locked contraction of the bladder, indicating that activity of these neurons alone is sufficient to trigger bladder contraction. Furthermore, micturating events were triggered with ChR2 stimulations, suggesting that activity of PMC Crh+ neurons is sufficient to trigger both contraction of the detrusor muscle in the bladder wall and relaxation of the urinary sphincters. In addition, although not analyzed here, additional effects of ChR2 stimulation, such as inducing defecation, were occasionally observed, suggesting functional heterogeneity within the PMC Crh+ neurons.

An Integration Center for Micturition Behavior

If PMC, and specifically Crh+ neurons of the PMC, mediates complex behavioral control over micturition, they must receive signals from higher brain areas, including those that process olfactory and social hierarchical information. We find that candidate inputs areas to Crh+ PMC neurons encompass all those previously described to project to the PMC region (Valentino et al., 1999). However, we find additional inputs such as dense projections from cortex (including somatosensory cortex) and motor-related superior colliculus.

Furthermore, whole-brain analysis reveals a widespread micturition regulatory network of at least ~3500–40000 neurons, indicating a high degree of convergence onto a maximum of ~500 Crh+ PMC neurons. The distribution of candidate presynaptic neurons was similar across animals and of three main classes: olfactory relay nuclei, cerebral cortex, as well as hypothalamic and brainstem nuclei. Olfactory cues are detected by main olfactory (volatile components) and vomeronasal (non-volatile) systems, which target distinct downstream circuits in the brain (Dulac and Wagner, 2006). Possible olfactory inputs to Crh+ neurons arise from main (BSTa, ventral pallidum) and vomeronasal (BSTp) systems, as well as from areas common to both systems (MPOA), suggesting an integration of information from both chemosensory pathways. Urine contains complex sensory signals that include both volatile and non-volatile components, and these findings might explain why mice with genetic ablation of vomeronasal systems have normal micturition pattern in response to female urine, but reduced micturition to male urine (Kaur et al., 2014; Leypold et al., 2002; Maruniak et al., 1986), as the former might rely mostly on the main olfactory system and the latter on the vomeronasal system. Potential shortest paths (Dulac and Torello, 2003; Kang et al., 2011) to reach PMC from the nose are: for main olfactory system, main olfactory bulb (MOB) ➔ medial amygdalar nucleus (MeA) ➔ BSTa/MPOA ➔ PMC; and for vomeronasal system, accessory olfactory bulb (AOB) ➔ BSTp/MPOA ➔ PMC. Crh+ PMC neurons also receive extensive projections from dorsal medial prefrontal cortex, including ACA and PL, regions that encode and causally affect social rank in rodents (Holson, 1986; Wang et al., 2011).

This complex and multi-modal set of projections to Crh+ PMC neurons reaffirms that they likely constitute the control center that integrates olfactory and social hierarchical information to regulate micturition. Coupled with our findings that PMC Crh+ neurons are direct regulators of micturition, these results provide an anatomical and molecular entry point into dissecting the decision-making process for context-dependent micturition behavior. The whole-brain survey of candidate input regions to PMC Crh+ neurons reveals inputs from areas of unclear function and may illuminate additional factors that regulate micturition, as well as inform possible mechanisms for bladder dysfunction in diseases of the central nervous system.

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Table S1

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Methods and Resources

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