The micturition reflex undergoes marked changes during prenatal and postnatal development but the mechanisms underlying these changes are largely unknown. As the CNS matures during the postnatal period, reflex voiding is brought under voluntary control involving higher brain centers (Capek and Jelinek, 1956; Sugaya et al., 1997; Sillen, 2001; Ng et al., 2007). Injuries of the adult nervous system including spinal cord injury (SCI) can lead to the reemergence of a loss of voluntary control (de Groat et al., 1998). Micturition in neonates of many species is dependent on activation of a spinal reflex pathway triggered when the mother licks the perineal region of the newborn (Capek and Jelinek, 1956; Sugaya et al., 1997; Sillen, 2001; Ng et al., 2007). This reflex pathway is essential to prevent urinary retention and consists of a somatic afferent limb in the pudendal nerve and a parasympathetic efferent limb in the pelvic nerve (de Groat et al., 1998). Human infants have a similar reflex pathway (Sillen, 2001). As the neonate develops, the perineal-to-bladder reflex weakens and is replaced by an inhibitory perineal-to-bladder reflex and the adult form of voiding (Fowler et al., 2008; de Groat and Yoshimura, 2015; de Groat et al., 2015). As the nervous system continues to mature, the spinal micturition reflex is gradually replaced by a spinobulbospinal reflex pathway activated by mechanosensitive afferent nerve activity to evoke micturition beginning in the rat between postnatal (P)16 and P18 (Fowler et al., 2008; de Groat and Yoshimura, 2015; de Groat et al., 2015).

The urinary bladder has two main functions; it must be able to expand to accommodate urine continuously produced by the kidneys and empty rapidly when needed. Storage and elimination functions involve the reciprocal functions of the bladder, urethra and external urethral sphincter, which are controlled by the coordination of the different tissue layers in the bladder wall and organized by complex neural pathways organized in the CNS and PNS (Andersson, 2002, 2004; Merrill et al., 2016). To sustain continuous storage and elimination phases, the urinary bladder is organized into three well-defined layers: the mucosa, muscularis propria, and the adventitia/serosa. The mucosal layer consists of transitional epithelial cells that line the lumen of the bladder and a lamina propria beneath the basement membrane of the epithelial cells (Andersson, 2002, 2004; Merrill et al., 2016). The transitional epithelial cells, termed the urothelium, function not only as an impermeable, nonadherent barrier, but also as a sensory component that is capable of responding to multiple and diverse mechanical and chemical stimuli. The urothelium can also respond to stimuli and release various factors including ATP, acetylcholine and nitric oxide (Birder and Andersson, 2013; Merrill et al., 2016). The barrier and signaling functions of the urothelium can be compromised during injury or inflammation, allowing toxic substances to reach the subepithelial nerve plexus and muscular layers, contributing to urinary urgency, frequency, and pain during voiding. The lamina propria lies between the urothelium and the detrusor and is composed of loose connective tissue, interstitial cells (ICs), vasculature, lymphatic vessels, nerve fibers and nerve terminals and may serve to integrate epithelial and smooth muscle input to maintain normal bladder function (Andersson, 2002, 2004; Birder and Andersson, 2013). The distribution and proximity of structural components of the urinary bladder suggests that reciprocal communication is possible between urothelial cells, components in the lamina propria and detrusor smooth muscles (Birder and Andersson, 2013).

ICs are found in many different tissues but, their role is poorly understood in the urinary bladder. In the gut, the role of a specialized class of ICs, the interstitial cells of Cajal (ICC) has been extensively studied and found to be key to generating slow waves and coordinating motility as well as regulating neurotransmission (Gfroerer and Rolle, 2013). In urinary bladder, despite numerous studies, the role of ICs is unknown. Antibodies against kit (proto-oncogene receptor tyrosine kinase, c-kit) labels ICC in the gut as well as macrophages (Ward et al., 1999). McCloskey (2010) showed kit-positive cells were located in both the detrusor and lamina propria of the mouse bladder (McCloskey, 2010) that had stellate-shaped morphology with numerous branches and containing gap junctions (Sui et al., 2002; McCloskey, 2010). In adult and neonatal rats as well as humans, lamina propria kit-positive cells are extensively linked by connexin (Cx)43 gap junctions to form a syncytium (Sui et al., 2002; Ikeda et al., 2007).

ICs in the lamina propria are sometimes called myofibroblasts based on their ultrastructural characteristics (Wiseman et al., 2003) and may interact with an extensive network of nerve fibers (subepithelial plexus), including afferents, that course through the lamina propria (Gabella and Davis, 1998). Immunohistochemistry as well as electron microscopy indicates a close association between lamina propria cells and nerve fibers (Wiseman et al., 2003; Davidson and McCloskey, 2005; Andersson and McCloskey, 2014). A subpopulation of kit-positive lamina propria cells that express platelet-derived growth factor receptor-α (PDGFRα) were also found in murine bladder (Koh et al., 2012). PDGFRα cells exhibited stellate or spindle-shaped morphology and formed a dense network in the lamina propria (Koh et al., 2012). Although the lamina propria is made up of a heterogeneous population of cells types certain cells may be important modulators of neural activity and form a communication link between the urothelium and detrusor; the importance of which may depend upon age or presence of pathology.

Several transient receptor potential (TRP) channels have been identified in the urinary bladder (Birder and Andersson, 2013; Merrill et al., 2016). These channels comprise a superfamily of non-specific cation channels that are generally, but variably, permeable to Ca²⁺, Na⁺, and K⁺ ions and may act as sensors of stretch and/or chemical irritation in the lower urinary tract. The TRP vanilloid (V) 4 (TRPV4) channel is expressed in different cells of the urinary bladder and allows Na⁺ and Ca²⁺ influx into the cell. Measurements of ionic currents and Ca²⁺ events induced by agonists (4α-PDD, GSK1016790A) or stretch have demonstrated functional expression of TRPV4 in urothelial cells (Gevaert et al., 2007b; Everaerts et al., 2010a,b,c) and detrusor (Thorneloe et al., 2008). TRPV4-KO mice demonstrate an abnormal voiding pattern and fewer voiding contractions compared to controls (Gevaert et al., 2007b) suggesting that TRPV4 channels contribute to normal bladder function as well as bladder pathology (Merrill and Vizzard, 2014; Merrill et al., 2016).

In this study, we identified a heterogeneous network of cells at the urothelial-lamina propria interface of rat pups with diverse morphology and numerous processes that exhibit PDGFRα- and TRPV4-immunoreactivity. Cells in this lamina propria network express spontaneous Ca²⁺ events mediated through the release of Ca²⁺ from internal stores and also respond to TRPV4 agonists with changes in Ca²⁺ signaling. Application of exogenous ATP evoked Ca²⁺ waves that propagate through the lamina propria cell network demonstrating a functional syncytium that may provide a critical communication link between the urothelium and the detrusor smooth muscle to convey sensory information or to affect detrusor contractility.

The current studies confirm the presence of a heterogeneous cellular network in the lamina propria that exhibits spontaneous Ca²⁺ transients that can be loosely or tightly coupled (wavefronts) between cells. In addition, these studies demonstrate several novel findings, including: (i) the predominance PDGFRα- and TRPV4-immunoreactivity in the lamina propria layer from early postnatal rat pups (P ≤ 21), (ii) ATP and a TRPV4 agonist activated and increased the number of lamina propria cells that exhibited active Ca²⁺ events, (iii) lamina propria cell activity was not random, with spatio-temporal maps and PTCL analysis demonstrating varying degrees of coupling (e.g., tight, partial or loose organization) and (iv) the coupling of Ca²⁺ activity of cells in laminar propria network could be modified to generate organized, propagating bands of activity (wave fronts) with ATP. These findings are consistent with the hypothesis that lamina propria Ca²⁺ signaling facilitates communication through this syncytial network to other cell types or tissue layers of the urinary bladder; our study shows the spatio-temporal patterning of this potential communication that may affect sensory processing and/or detrusor contractility.

The spinobulbospinal micturition reflex is triggered by tension receptor afferents in the bladder and begins to elicit voiding in the rat between P16-18 (Capek and Jelinek, 1956; Girard et al., 2016) and continues to mature during the postnatal weeks 3–6 (Capek and Jelinek, 1956; Ng et al., 2007). As the CNS matures during the postnatal period in humans, reflex voiding becomes voluntary (2–5 years) and originates in the higher brain centers (de Groat et al., 1998; de Groat and Araki, 1999; Zvarova and Zvara, 2012). Studies in rats (Sugaya et al., 1997) and human infants (Sillen, 2001) demonstrate that supraspinal circuits involved in mature micturition reflex patterns exist in neonatal animals and infants but may not be functional or may function in an inhibitory manner prior to the emergence of a functional, adult micturition pattern (de Groat and Araki, 1999). The micturition reflex continues to mature postnatally to achieve the adult form of reflex voiding. The perineal-to-bladder reflex becomes progressively weaker during the postnatal period and replaced by an inhibitory perineal-to-bladder reflex and the adult micturition reflex. It is hypothesized that upregulation of bulbospinal projections as well as synaptic remodeling in the sacral parasympathetic nucleus in the lumbosacral spinal cord and in the brain contribute to the down-regulation of the perineal-to-bladder reflex. This may be due to competition between supraspinal and segmental neural inputs (Araki, 1994; Araki and de Groat, 1997; de Groat and Araki, 1999).

To function efficiently, the urinary bladder must relax during filling and contract forcefully during micturition. Therefore, communication between the tissue layers in the bladder wall is critical for normal bladder function. Located between the urothelium and detrusor, the lamina propria is frequently described as a functional syncytium and is ideally located to facilitate communication between these tissue layers. Ca²⁺ signals have previously been identified using Ca²⁺ sensitive dyes in isolated lamina propria cells (Wu et al., 2004) and with in situ preparations (Gray et al., 2013). Ca²⁺ transients can also be evoked by electrical stimulation (Gray et al., 2013) or with purinergic agonists (Wu et al., 2004). In our study using tissue wholemounts, Ca²⁺ events in the rat pup lamina propria cells had long durations with slow upstroke and downstroke phases, and had a frequency similar to those previously described in the guinea pig (Gray et al., 2013). These Ca²⁺ events were largely dependent on Ca²⁺ release from internal stores because CPA significantly reduced the overall duration of cell activation and the number of cells exhibiting Ca²⁺ events in the lamina propria network. Propagation of Ca²⁺ transients through the lamina propria was first described using a transverse bladder preparation and a photodiode array with a circumscribed sensing area (0.95 × 0.95 mm) for each photodiode. With this system, mechanical or carbachol induced Ca²⁺ transients were found to originate near the urothelial-suburothelial interface with spread to the detrusor (Kanai et al., 2007). This relatively low-resolution system provided evidence that Ca²⁺ transients can propagate through the lamina propria. Using higher resolution, Ca²⁺ spreading between two lamina propria cells via a connecting process was detailed in guinea pig (Gray et al., 2013). In lamina propria cells from rat pup tissue wholemounts, we found Ca²⁺ events often spreading along processes between cells. The ability of the lamina propria network to have a range of Ca²⁺ behaviors ranging from ongoing, loosely-coupled individual cellular activity to where cells were activated nearly synchronously in a particular direction to form a Ca²⁺ wavefront that propagated substantial distances throughout the lamina propria is indicative of flexible coupling between cells in this region (see Figures 3, 7). Although the mechanism underlying the formation of a Ca²⁺ wavefront is unclear, rat pup lamina propria have a high concentration of Cx 43 consistent with the presence of gap junctions (Ikeda et al., 2007). Intercellular Ca²⁺ waves have been observed to propagate at speeds of ~10–20 μm/s, somewhat slower than the 60–70 μm/s we observed in rat pup lamina propria (Leybaert and Sanderson, 2012).

There is increasing evidence that stretch or chemical stimuli of the urothelium initiates the production and release of factors and transmitters including ATP, ACH, and nitric oxide (Birder et al., 1998; Yoshida et al., 2006). ATP (that is not degraded) can stimulate autocrine or paracrine pathways that may convey sensory information to the CNS (Schwiebert and Zsembery, 2003). The transduction pathways within the urinary bladder are affected by receptor subtype expression and their proximity to the urothelium. The tissues and cell types that may contribute to purinergic signaling include nerves in the suburothelial nerve plexus, smooth muscle cells, lamina propria cells including ICs as well as inflammatory cells (Birder, 2005; Birder and Andersson, 2013; Li et al., 2013). For example, ATP was found to activate inward currents (Wu et al., 2004) and Ca²⁺ transients in freshly isolated (Sui et al., 2004) or cultured suburothelial myofibroblasts from human bladder (Cheng et al., 2011). Kit-positive ICs express P2X and P2Y (i.e., P2X3 P2Y2, P2Y4, and P2Y6) receptors and are proposed to form a functional syncytium with smooth muscle cells (Sui et al., 2006; Drumm et al., 2014). In response to ATP, ICCs in the gastrointestinal tract generate P2Y-dependent intracellular Ca²⁺ transients that may then propagate to smooth muscle cells via gap junctions to alter contractility (Drumm et al., 2014). Although the mechanism coupling ICs to sensory activity is unknown, the location of ICs and the responsiveness to ATP suggest they may have a regulatory role in the afferent limb of the micturition reflex (Wu et al., 2004). In the present study, exogenous ATP increased Ca²⁺ transient activity in numerous lamina propria cells that culminated in multiple Ca²⁺ waves propagating through the lamina propria. In addition, these studies demonstrate that exogenous ATP can convert a loosely connected behavior of lamina propria cells to a highly organized behavior with the formation of a Ca²⁺ wavefront. This finding is consistent with the hypothesis that ATP released from urothelium or other tissues (i.e., suburothelial nerves) increases lamina propria Ca²⁺ signaling and facilitates communication through this syncytial network to other cell types or tissue layers (Andersson and McCloskey, 2014). Having a large area of the lamina propria network undergoing near-synchronous activity may provide a much more potent signaling/stimulus to other cell types compared to loosely-coupled patterns of activation of these cells. In addition to the lamina propria network acting as an amplifier of signals/stimuli (Andersson and McCloskey, 2014), we suggest that the lamina propria network is a primary source or originator of coordinated activation of the urinary bladder where it senses and responds to stimuli and communicates this information to the whole urinary bladder. Such a signaling network would be important when neural circuits are not fully mature (i.e., postnatal development) or when compromised by neural injury or disease.

Multiple TRP channels from different subfamilies are expressed in the urinary bladder and have specific tissue distributions in the LUT. These channels are activated by numerous exogenous and endogenous mediators (Skryma et al., 2011; Deruyver et al., 2015) and may have functional roles in the micturition reflex (Andersson et al., 2010; Merrill et al., 2016). Many of these channels are also implicated in bladder disorders including overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC) (Gevaert et al., 2007a,b; Nilius et al., 2007). Recent studies suggest the involvement of TRPV4 in both normal urinary bladder function and dysfunction (Andersson et al., 2010). TRPV4 was first demonstrated in basal and intermediate urothelial cells (Gevaert et al., 2007a,b) and confirmed by many other studies (Merrill et al., 2016). The functional expression of TRPV4 in urothelial cells has been established following measurements of ionic currents and Ca²⁺ events induced by agonists (4 alpha-phorbol 12,13-didecanoate (4α-PDD), GSK1016790A) or stretch (Kullmann et al., 2009; Mochizuki et al., 2009; Xu et al., 2009). TRPV4 is also expressed in the detrusor smooth muscle; however, transcript levels were found to be approximately 20- to 36-fold higher in the urothelium compared to detrusor smooth muscle (Thorneloe et al., 2008; Xu et al., 2009; Merrill et al., 2012). TRPV4 expression has also been examined in DRG neurons innervating viscera (Yamada et al., 2009; Merrill et al., 2012; Alexander et al., 2013; Girard et al., 2013) but functional evidence is lacking (Alexander et al., 2013). In contrast, the present studies demonstrate functional expression of TRPV4 in lamina propria cells in postnatal rat pups. In wholemount tissue preparations isolated from rat pups aged ≤ P21, TRPV4-IR was observed in cells in the lamina propria that exhibited similar morphology to those expressing PDGFRα-IR. Application of the TRPV4 agonist, GSK1016790, increased the time lamina propria cells were active and increased the number of cells that exhibited active Ca²⁺ events as evidenced by the rate of integrated Ca²⁺ activity. The failure of the TRPV4 antagonist, GSK2193874, to alter the number of lamina propria cells displaying Ca²⁺ transients or the duration of Ca²⁺ transients may indicate that Ca²⁺ influx through TRPV4 channels does not contribute to basal Ca²⁺ signaling in the lamina propria.

The genetic or pharmacological manipulation of TRPV4 has helped to elucidate its physiological role in the micturition reflex. TRPV4 knockout mice exhibited abnormal urine voiding patterns characterized by decreased frequency of voiding contractions and increased frequency of nonvoiding contractions, longer intermicturition intervals and increased total urine volume per void (Gevaert et al., 2007b; Everaerts et al., 2010c). Administration of the TRPV4 agonist, 4α-PDD, to conscious rats resulted in an increase in the amplitude of reflex bladder contractions during cystometry (Birder, 2007). GSK1016790A, a highly selective TRPV4 agonist that is ~300-fold more potent than 4α-PDD, similarly induced bladder hyperactivity in vivo in mice (Thorneloe et al., 2008) and rats (Aizawa et al., 2012). Systemic administration of the selective and potent TRPV4 antagonist, HC-067047, decreased voiding frequency and increased bladder capacity in mice and rats following CYP-induced cystitis (Everaerts et al., 2010c) or repeated variate stress (Merrill and Vizzard, 2014). We suggest that the bladder sensory roles of TRPV4 in the normal micturition reflex or following injury or pathology may also be related to TRPV4 expression and function in lamina propria cells.

At the urothelial-lamina propria junction, the network of cells expressing PDGFRα- or TRPV4-IR was sparse and calcium events were not observed. In wholemount preparations from rat pups aged ≤ P21, we consistently identified a dense network of PDGFRα- or TRPV-IR cells in the lamina propria. The reasons underlying these observations are unknown but may reflect the continuing maturation of the micturition reflexes during the early postnatal period. It has previously been speculated that the functional syncytium between ICs and the detrusor smooth muscle is important for coordination of bladder emptying in the early postnatal period because of the absence of mature and functional neural input needed to coordinate the activities of the CNS and PNS including the urinary bladder (Kanai et al., 2007). The current studies in rat pups aged ≤ P21 are consistent with this suggestion as demonstrated by: (i) the predominance of the lamina propria cellular network in early postnatal rat pups; (ii) the ability of ATP and a TRPV4 agonist to activate and increase the number of lamina propria cells that exhibited active Ca²⁺ events; and (iii) the ability of ATP and TRPV4 agonist to increase the rate of integrated Ca²⁺ activity corresponding to coupled lamina propria network events and the formation of propagating wavefronts. Thus, the lamina propria network may have an active role in sensing (e.g., distension) and signaling, perhaps reciprocally, between bladder layers and cell types to achieve coordinated bladder function. The importance of the lamina propria network may depend upon the integrity and maturity of neural pathways that coordinate micturition reflex events.

Altered ATP purinergic signaling in the LUT that include changes in ATP release, expression or density of purinergic receptors and/or expression of ATPases and other ecto-nucleotidases, may contribute to voiding dysfunction, mechanical hypersensitivity and pain (Sun et al., 2001a,b). Increased levels of urinary ATP have been demonstrated in patients with IC (Sun et al., 2001a,b) and overactive bladder (Silva-Ramos et al., 2013; Burnstock, 2014). Primary bladder urothelial cells from these patients also exhibit increased ATP release in response to a variety of stimuli (Sun et al., 2001a; Sun and Chai, 2006). Changes in P2X receptor subtype expression in various bladder tissues have also been demonstrated in patients with IC (Sun and Chai, 2004), detrusor instability (O'Reilly et al., 2002) or bladder outlet obstruction (O'Reilly et al., 2001). In addition, altered protein and transcript expression of TRPV4 in the urinary bladder has been demonstrated during postnatal development and in mice with chronic, urothelial overexpression of NGF (Merrill et al., 2012; Girard et al., 2013). TRPV4 antagonists decrease bladder activity making it a promising target for overactive bladder and other bladder disorders (Birder, 2007; Gevaert et al., 2007b; Merrill et al., 2016). In future studies, it would be of interest to examine the lamina propria network in wholemount preparations from preclinical animal models of bladder dysfunction or SCI (i.e., upper motoneuron injury) to determine if the presence of the lamina propria network at the urothelial-lamina propria junction is changed and/or if spontaneous and evoked Ca²⁺ transients and network activity is altered. A change in the number of cells exhibiting Ca²⁺ events, their pattern of activation, the duration of Ca²⁺ events and/or the ability of mediators (e.g., ATP, TRPV4 agonists) to increase the rate of integrated Ca²⁺ activity corresponding to coupled lamina propria network events could contribute to altered sensory processing (e.g., mechanosensation, pain) and altered urinary bladder function (i.e., hyperactivity). There is precedent for plasticity in the expression of lamina propria cellular network. ICs in the bladder wall following SCI (5 weeks post injury) are decreased, target organ innervation is reduced and the smooth muscle is hypertrophied (Johnston et al., 2012). However, there are differing reports concerning changes in ICs from individuals with bladder dysfunction (e.g., neurogenic and idiopathic detrusor overactivity; McCloskey, 2010). Thus, it would be of interest and potential importance to continue to examine the lamina propria network at the urothelial-lamina propria junction following neural injury, disease or urinary bladder dysfunction to fully understand its functional significance.

The studies described from the Vizzard laboratory were performed in accordance with institutional and national guidelines and regulations. The University of Vermont Institutional Animal Care and Use Committee approved all experimental protocols involving animal use. Animal care was under the supervision of the University of Vermont's Office of Animal Care Management in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress or distress.

Analyzed the data, performed experiments, conceived, discussed and outlined the experimental design: TH, GH, and MV. Wrote the paper, drafted and revised paper: TH, GH, MN, and MV.