Keywords

Introduction

Micturition Reflex Pathways to the Urogenital Tract

Micturition is organized between two modes of operation: storage and elimination. During storage, somatosympathetic excitatory inputs to the urethral sphincters and sympathetic inputs to the bladder wall are tonically active [1, 2]. In contrast, during reflexive or voluntary elimination, parasympathetic inputs to the urinary bladder wall are active whereas somatosympathetic inputs to the bladder wall and urethral sphincters are inhibited [3]. Although spinal reflexes underlie most of the storage phase, reflexive or voluntary micturition reflex mechanisms are modulated by supraspinal regulation in the pontine micturition center [4].

Slowly adapting mechanoreceptors in the urinary bladder wall underlie the switch from storage to elimination [5]. The thinly myelinated Aδ afferent fibers of the hypogastric and pelvic nerves increase their activity as hydrostatic pressure rises [6]. Bladder afferent nerves that terminate peripherally in the urinary bladder may also signal through unmyelinated C-fibers that respond to nociceptive stimulation by chemicals, inflammation, and elevated intravesical pressures [7–9]. Although C-fibers are quiescent during normal bladder filling, their activation may contribute to the development of lower urinary tract (LUT) symptoms and functional disorders of the urinary bladder [10, 11].

Bladder afferent fibers from the pelvic nerve project into Lissauer’s tract where collateral branches extend along the superficial laminae of the dorsal horn (DH) [2, 8, 12] (Fig. 19.1). The ventromedial collateral branch follows the medial edge of the DH (i.e., medial collateral pathways) into the dorsal commissure (DCM) and receives inputs from the pudendal nerve and urogenital structures [2, 8, 12]. The ventrolateral collateral branch (i.e., lateral collateral pathway, LCP) projects on the lateral edge (lamina I) of the DH into the sacral parasympathetic nucleus (SPN) that contains preganglionic parasympathetic neurons projecting to the periphery [2, 8, 12] (Fig. 19.1). In addition to synapsing directly on preganglionic parasympathetic neurons in some species, primary bladder afferent fibers also synapse on interneurons in the lumbosacral DCM, superficial DH, and SPN [2, 6]. These interneurons project locally in the spinal cord or to supraspinal cortical modulatory centers and are important in the normal micturition reflex [2, 6].

Fig. 19.1
figure 1

Wiring diagram of micturition reflexes emphasizing the reflex elements that express PACAP-IR. The PACAP/receptor system has been identified in micturition reflex pathways with contributions to normal LUT function as well as that after neural injury, disease, or inflammation. PACAP-IR is expressed in normal LUT pathways but expression is dramatically increased following injury, disease, and inflammation of the urinary bladder (see text for details). Robust PACAP-IR is expressed in lumbosacral DRG (2), spinal cord (1), including the superficial laminae of the DH (1) and the LCP of Lissauer’s tract (white arrows, 1). Bladder afferent cells in the DRG, retrogradely labeled with the conventional tracer, Fast Blue (FB), express PACAP-IR (white arrows, 2). Not all PACAP-IR cells in the lumbosacral DRG are bladder afferent cells (blue arrows, 2). Not all presumptive bladder afferent cells expressing FB, also exhibit PACAP-IR (yellow arrow, 2). PACAP-IR is also present in the urinary bladder including expression in urothelial cells that line the urinary bladder and in nerve fibers of the suburothelial nerve plexus (3). Postganglionic neurons in the major pelvic ganglia (MPG) also express PACAP-IR (not shown). INT interneurons, PGN preganglionic neurons, SPN sacral parasympathetic nucleus, CC central canal, PMC pontine micturition center, EUS external urethral sphincter

Full size image

Urothelial Signaling

The urothelium lines the bladder mucosa and responds to mechanical, chemical, and thermal stimuli [13]. In response to these stimuli, urothelial cells secrete factors like urinary proteins and signaling molecules suggesting a role in urinary bladder sensory transduction [13, 14]. Urothelial cells also express receptors and mechanosensitive channels to respond to the extracellular environment [15–18]. Given that the urothelium may have a sensory influence on micturition reflex function, any disruption to urothelial signaling mechanisms and/or the underlying neural network may contribute to pathological conditions of the urinary bladder [13].

Neurochemistry of Micturition Pathways

Bladder afferent fibers contain a variety of neuropeptides, including calcitonin-gene related peptide (CGRP), substance P (SP), neurokinin A, neurokinin B, vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), cholecystokinin, and enkephalins [12, 19–24] (Fig. 19.1). With the exception of CGRP, all of these substances are predominantly expressed in small diameter (presumably C-fiber) afferents [12, 19, 20, 25–32]. The administration of capsaicin, which acts selectively on small-diameter afferents to deplete neurotransmitter stores, reduces the levels of SP, neurokinin A, and CGRP but not VIP or enkephalin within the pelvic viscera [33]. These findings are consistent with SP, CGRP, and related tachykinins expression in afferent pathways to the pelvic viscera [33]. The following sections focus on the expression, distribution, and functional plasticity of members of the VIP–secretin–glucagon family of hormones, PACAP and VIP in micturition reflex pathways (Fig. 19.1). The contributions of other peptides to micturition reflex pathways have recently been described [24].

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and VPAC/PAC1 Receptor Signaling

PACAP belongs to the VIP/secretin/glucagon family of bioactive peptides and was isolated from hypothalami based on its stimulation of anterior pituitary adenylyl cyclase (AC) activity [34, 35]. The rat PACAP precursor protein consists of 175 amino acid residues with posttranslational processing resulting in two ∝-amidated forms, PACAP38 and PACAP27 [34, 36–39]. PACAP38 has 38 amino acid residues, whereas PACAP27 has the carboxyl terminus truncated and exhibits 68 % homology to VIP [35, 37, 40]. The distribution of these two forms is tissue-specific with PACAP38 typically predominating expression in most tissues [34, 41]. PACAP38 remains identical among mammalian species suggesting similar physiologically important roles such as signaling modulation and trophic functions in the nervous and endocrine systems [34, 42].

There are three distinct G-protein-coupled receptors for PACAP and VIP: PAC1, VPAC1, and VPAC2 [43–48] (Fig. 19.2). PAC1 receptors exhibit high affinity for PACAP and display unique patterns of AC and phospholipase C (PLC) activation for PACAP27 or PACAP38 [48–52]. The potency of PACAP27 and PACAP38 to PAC1 receptors is affected by alternative splicing to receptor transcripts resulting in the presence (short) or absence (very-short) of a 21-residue insert into the amino-terminal extracellular domain [53] (Fig. 19.2). Other variants from the alternative splicing of two 84 base pair HIP and HOP cassettes result in the unique patterns of AC and PLC activation [48] (Fig. 19.2). VPAC1 and VPAC2 receptors, on the other hand, exhibit high affinity for both PACAP and VIP and are solely coupled to AC [48]. The expression of PAC1 and VPAC receptors is tissue- and cell type-specific. It was previously shown that rat superior cervical ganglia sympathetic neurons express PAC1(short)HOP1 while VPAC receptors were sparsely expressed in ganglion non-neuronal cells [49–51, 54–60].

Fig. 19.2
figure 2

LUT tissues express PAC1 receptor variants. Complementary DNA templates were prepared from rat S1 spinal cord, S1 DRG, and bladder detrusor and urothelium total RNA. The region spanning the alternative splice site for the HIP and HOP exons within the third cytoplasmic loop was amplified using PACAPR1/2 oligonucleotide primers. Six third cytoplasmic loop isoform fragments containing neither, one or both HIP and HOP cassettes can potentially be amplified with these primers. LUT tissues express PAC1 receptor isoforms in a tissue-specific manner. S1 DRG express predominantly the one cassette isoform; other tissues possess both the null and the one cassette variant. Schematic shading: Dark grey, short region containing exons 4 and 5; light grey, HIP exon cassette; black, HOP cassette. Thick line, region amplified using PACAPR1/2 primers. LUT tissue expression of PAC1 receptor isoforms also results from alternative splicing in amino-terminal extracellular domain. Complementary DNA templates from LUT samples described above were amplified using primers PACAPR3/4, which flank the amino-terminal extracellular domain splice site. The amplified fragments of indicated sizes represent isoforms with both (short) or neither (very short) exons 4 and 5. All LUT tissues express the short variant; urinary detrusor smooth muscle also demonstrates very short PAC1 receptor expression. Shading in schematic denotes alternatively spliced exons. Thick line, region amplified using primers PACAR3/4. Figure modified from ref. [98]

Full size image

PACAP and PAC1 Receptor Neuronal Functions in the LUT

PACAP peptides have diverse functions in endocrine, nervous, gastrointestinal, and cardiovascular systems and are expressed in many central nervous system neurons and sensory and autonomic ganglia [34, 36, 41, 42, 61–76]. PACAP facilitates neuronal calcium influx, induces depolarization of the membrane, activates AC and PLC, and stimulates neurotransmitter secretion [49, 50, 54, 55, 58–60, 77–79]. Widespread PACAP-immunoreactivity (IR) has been demonstrated in nerve fibers within the urinary bladder smooth muscle, suburothelial plexus and surrounding blood vessels [80] (Fig. 19.1). Neonatal capsaicin treatment significantly reduced PACAP suggesting these fibers are derived from sensory neurons [80]. These results are consistent with the expression of PACAP in DRG and its neurochemical plasticity following nerve injury or inflammation [42, 81–83] (Fig. 19.1).

PACAP- and VIP-Mediated Effects on Urothelium and Detrusor Smooth Muscle

The urothelium acts as a selective barrier to prevent urinary constituents from penetrating the underlying tissue [84, 85]. A disruption to the properties of barrier function may occur through trauma, infection or disorders affecting the bladder like bladder pain syndrome/interstitial cystitis (BPS/IC) or spinal cord injury (SCI) [86, 87]. It has been suggested that this loss of barrier integrity contributes to the altered sensory processing observed in cystitis. Recent studies have demonstrated that the urothelium expresses PAC1 receptors that upon stimulation release ATP to stimulate receptors on underlying sensory nerve fibers [88] (Fig. 19.2). ATP release was evoked by PACAP27, PACAP38, and VIP application to cultured urothelial cells with PACAP27 and PAC1 receptor antagonism blocking ATP release [88]. These results suggest PACAP and PAC1 signaling may regulate micturition reflex function at the level of the urothelium [88] (Figs. 19.1 and 19.2).

PACAP and VIP have direct effects on smooth muscle cells. PACAP or VIP elicit relaxation of guinea pig stomach, rat ileum, rabbit iris sphincter and dilator muscles, cat and human esophageal sphincter, and human and guinea pig airways but elicit contraction in guinea pig ileum and gall bladder [89]. The effects of PACAP on urinary bladder smooth muscle have not been well described despite PACAP, PAC1, and VPAC expression in nerve fibers within the detrusor [80] (Figs. 19.1 and 19.2). PACAP27 has a small effect on isolated bladder smooth muscle strips even though it facilitates micturition in conscious, open outlet rats [90]. These studies, however, did not take into account PAC1 and VPAC receptor cross talk and the peptide selectivity of various PAC1 isoforms.

Unlike PACAP, VIP is expressed in postganglionic efferent neurons of the major pelvic ganglia (MPG) and minimally innervates the urinary bladder [80, 91–93]. VIP administration to the detrusor smooth muscle had no effect on spontaneous or carbachol-induced bladder contractions but intrathecal or intra-arterial VIP administration facilitated micturition [94]. These conflicting roles may result from VIP receptor distribution varying across species and target tissue [94–97]. Taken together, it appears that PACAP/receptor signaling has more influence on micturition reflex function than VIP/receptor signaling [94–98].

PACAP and VIP Expression and Effects on MPG Neurons

PACAP/receptor expression has been demonstrated in the MPG, ganglia that supply autonomic (sympathetic and parasympathetic) innervation to the urinary bladder, following neuronal injury. PAC1, VPAC1, and VPAC2 transcripts were reported in the MPG in cell culture for 4 h [99], with only VPAC2 transcript significantly increased by day 3 [100]. PACAP transcript and PACAP-IR similarly increased in the MPG by day 3 in culture [100]. Unlike PACAP, VIP transcript expression remained unchanged in a 3-day culture [100]. Furthermore, the application of VIP, PACAP, and maxadilan, a PAC1-selective agonist, increased neuronal excitability and decreased after-hyperpolarization in the MPG [99]. Taken together, these studies suggest PACAP/receptor signaling in the MPG may have a role in micturition reflex function following injury (Fig. 19.1).

PACAP or VIP Knockout Mice Exhibit Altered Micturition Reflexes

Bladder dysfunction and altered somatic sensation have previously been demonstrated in mice with a genetic disruption or deletion to PACAP or VIP. PACAP (+/−) and PACAP (−/−) mice display less somatic sensitivity to mechanical stimuli in the pelvic and hindpaw regions relative to controls [101]. On the other hand, VIP (−/−) mice display increased somatic sensitivity in the pelvis and decreased paw pressure threshold following inflammation [102]. These dissimilarities may reflect distinct roles for VIP and PACAP in bladder sensory function. In contrast to the observed mechanosensitivity differences, both knockout mice exhibited an increase in bladder mass with hypertrophy specific to the lamina propria and detrusor smooth muscle in PACAP (−/−) mice and only the detrusor smooth muscle in VIP (−/−) mice [101, 103].

Functionally, PACAP (−/−) mice have increased bladder capacity, void volume, and longer intercontraction intervals relative to controls [101]. It has been argued that these mice exhibit partial outlet obstruction because of extended and incomplete emptying of the bladder [101]. VIP (−/−) mice, however, do not functionally present with changes in the basal tone of the bladder but do have increased void volume, longer intercontraction intervals, and complete emptying of the bladder [102]. Bladder dysfunction in VIP (−/−) mice may result from functional changes within the bladder itself or neuroplasticity among bladder afferent cells. Along with the increase in tissue mass, the urinary bladders of VIP (−/−) mice have increased urea permeability, increased basal expression of NGF and an exaggerated proinflammatory response to inflammation [102–104]. Additionally, there are elevated basal levels of phosphorylated cAMP response-element binding protein (pCREB) in lumbosacral (L1, L2, L5–S1) DRG and in L6 and S1 afferent neurons projecting directly from the bladder of VIP (−/−) mice suggesting elevated afferent activity of the urinary bladder [103].

Neuroplasticity of PACAP/Receptor Expression and Function with Cystitis

The regulation of transcript and peptide expression of PACAP and its receptors previously demonstrated with cyclophosphamide (CYP)-induced cystitis has been suggested to underlie the development of urinary bladder dysfunction. Following a downregulation in transcript expression after acute (4 h) CYP-induced cystitis, PACAP transcript expression is dramatically upregulated in the urothelium and L6 and S1 DRG after intermediate (48 h) or chronic (10 day) CYP-induced cystitis [88]. Similarly, transcript expression of the PAC1 receptor is down-regulated in the urothelium, detrusor smooth muscle and L6 and S1 DRG after acute (4 h) CYP-induced cystitis, but upregulated in the urothelium and detrusor smooth muscle after intermediate (48 h) or chronic (10 day) CYP-induced cystitis [88]. VPAC1 and VPAC2 transcript expression, however, remains upregulated in the urothelium and detrusor smooth muscle from acute (4 h) CYP-induced cystitis to intermediate (48 h) CYP-induced cystitis with the down-regulation of VPAC2 transcript expression occurring after chronic (10 day) CYP-induced cystitis [88]. PACAP-IR in the spinal cord is restricted to nerve fibers and is increased in micturition reflex associated regions after intermediate (48 h) or chronic (10 day) CYP-induced cystitis [22, 105]. Following intermediate or chronic CYP-induced cystitis, PACAP-IR is increased in the superficial laminae (I-II) of the DH, medial to lateral extent of the dorsal horn, LCP of Lissauer, SPN, and S1 spinal segments [22, 105] (Fig. 19.1). Additionally, the percentage of Fast Blue labeled bladder afferent cells positive for PACAP increased in L1-L2, L6, and S1 DRG following chronic (10 day) CYP-induced cystitis [22] (Fig. 19.1).

The aforementioned regulation of PACAP and its receptors in areas associated with the micturition reflex suggests this neuropeptide may have a role in bladder dysfunction with inflammation (Fig. 19.2). In support of this, the intrathecal (L6-S1) or intravesical administration of a PAC1 receptor antagonist, PACAP6-38, was able to increase bladder capacity but not intravesical pressure with intermediate (48 h) CYP-induced cystitis [98]. The different routes of administration with similar functional effects suggest PACAP6-38 may have multiple sites of action. Administration of PACAP6-38 at the level of the spinal cord may be acting on superficial DH neurons to block PACAP release from C-fiber afferents, whereas, PACAP at the level of the urinary bladder may be acting on urothelial, suburothelial or detrusor smooth muscle cells [98] (Fig. 19.1). Despite not yet knowing its specific site of action, the inhibition of aberrant PACAP signaling seems to be a promising target to reduce voiding frequency with cystitis.

PACAP Expression in LUT with CYP-Induced Cystitis in PACAP Promoter-Dependent EGFP BAC Transgenic Mice

We previously demonstrated an upregulation of PACAP expression in rodent micturition pathways following CYP-induced cystitis [98]. We subsequently examined the effects of CYP-induced cystitis (4 h, 48 h, chronic) in PACAP promoter-dependent EGFP BAC transgenic mice [106]. We induced bladder inflammation in adult mice by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) cystitis. In control (no inflammation) animals, low basal expression of PACAP-EGFP+ fibers was present in the superficial DH at all segmental levels examined (L1, L2, L4–S1). Dorsal root ganglia (DRG; L1, L2, L6, S1) from control animals also exhibited PACAP-EGFP+ cells. After CYP-induced cystitis, PACAP-EGFP+ cells increased dramatically in spinal segments and DRG (L1, L2, L6, and S1) involved in micturition reflexes. Small diameter, PACAP-EGFP+ DRG cells co-localized with TRPV1- and TRPV4-IR [106]. The density of PACAP-EGFP+ nerve fibers was increased in the superficial laminae (I–II) of the L1, L2, L6, and S1 DH. No changes in PACAP-EGFP+ nerve fibers were observed in the L4–L5 segments. PACAP-EGFP+ nerve fibers also increased in the lateral collateral pathway in L6–S1 spinal cord. Following CYP-induced cystitis, PACAP-EGFP+ urothelial cells were observed and the number of PACAP-EGFP+ urothelial cells increased with duration of cystitis. PACAP-EGFP+ urothelial cells were co-localized with TRPV4-IR [106]. Changes in PACAP expression in LUT pathways after cystitis may play a role in altered visceral sensation (allodynia) and/or increased voiding frequency in the chronic inflammatory pain syndrome, interstitial cystitis/bladder pain syndrome.

Neuroplasticity of PACAP/Receptor Expression and Function with Spinal Cord Injury (SCI)

SCI has been demonstrated to regulate the transcript and peptide expression of PACAP and its receptors within the spinal cord and urinary bladder. An increase in PACAP and PAC1 receptor transcript expression is observed in the spinal cord following a moderate compression model of SCI [107]. PACAP- and PAC1-immunoreactive cells are also increased around the site of injury and co-localized with NeuN-positive cells (i.e., neuronal marker) [107]. In other studies utilizing spinal cord (Thoracic (T)7–T9) transection, PACAP-IR is increased 6 weeks after SCI in micturition reflex associated regions [108]. Within the upper lumbar (L1–L2) spinal cord, PACAP-IR increased in the superficial laminae (I–II) of the DH, medial to lateral extent of the DH, and a fiber bundle extending laterally from Lissauer’s tract [108]. Similarly within the lumbosacral (L6–S1) spinal cord, PACAP-IR increased in the DH, DCM, SPN, and LCP of Lissauer [108]. Increased PACAP-IR is not limited to the spinal cord, but the percentage of PACAP-positive bladder afferent cells labeled with Fast Blue are also increased in L1–L2, L6, and S1 DRG 48 h to 6 weeks after SCI [108]. Unlike the increased PACAP-IR observed at the level of the spinal cord and DRG, the urinary bladder has decreased PACAP-IR in the urothelium and detrusor smooth muscle from 5 days to 3 weeks after SCI [108].

The regulation of PACAP and its receptors around the site of SCI and micturition reflex regions suggests this neuropeptide may have a protective role to help facilitate bladder function. In support of this, the intrathecal administration of PACAP-38 following transection of the T8–T9 spinal cord resulted in large amplitude and long duration bladder contractions under isovolumetric conditions [109]. Additionally, intrathecal administration of a PAC1 receptor antagonist, PACAP6-38, following transection of the T8–T10 spinal cord reduced filling, threshold and peak micturition pressures, number and amplitude of non-voiding contractions, and had shorter intercontraction intervals [110]. Taken together, these studies suggest PACAP may act on parasympathetic efferent pathways at the level of the spinal cord and/or DRG [109]. Aside from its possible role in bladder function, PACAP may also be protecting from a loss of motor function. PACAP (+/−) mice showed a greater injury volume surrounding SCI and also exhibited lower Basso Mouse Scale motor scores on days 3, 7, and 14 suggesting impaired motor function [107]. These studies demonstrate the significance of PACAP regulation following SCI and argue for a role of PACAP in both bladder and somatomotor function with injury.

Role of Nerve Growth Factor (NGF) and Associated Receptors in LUT Plasticity

Cytokines and growth factors, including NGF, are upregulated at the site of tissue injury, inflammation, and/or target organ hypertrophy [111–115]. Following noxious peripheral stimulation, for example, levels of neuroactive compounds (e.g., enkephalin [112], dynorphin [116], CGRP [115, 117, 118], SP [23, 112, 116, 118], neuropeptide Y [112]; neuronal nitric oxide synthase (nNOS) [28, 119, 120] and PACAP [22, 121]) have been demonstrated to increase in DRG and spinal cord neurons. NGF, in particular, is also released from the target organ for tyrosine kinase receptor (Trk) type 1 (TrkA) binding and retrograde transport in DRG afferent neurons [122]. The subsequent increase in NGF expression within the DRG neurons may induce increased production of neuropeptides (i.e., SP, CGRP, and PACAP) and alter sensory transduction [115, 117, 118]. In addition, a large percentage of pelvic visceral afferent neurons express neurotrophic factor receptors, including Trk for NGF and related substances [123–126]. Following cystitis or SCI, neurotrophic factor receptors exhibit neuroplastic increases in TrkA- and TrkB-IR and Trk phosphorylation in bladder afferent neurons [125, 126].

CYP-Induced Cystitis

Increases in the number of TrkA-immunoreactive cell profiles were detected in the L1 and L6 DRG (fourfold) and the S1 DRG (1.5-fold) but not in the L2, L4, and L5 DRG with CYP-induced cystitis of acute and chronic duration compared with control rats [125]. The number of TrkB-IR cell profiles increased in the L1 and L2 DRG (L1: 2.6-fold; L2: 1.4-fold) and in the L6 and S1 DRG (L6: 2.2-fold; S1: 1.3-fold) only after acute CYP treatment (8 h) [125]. After CYP treatment, the percentage of bladder afferent cell profiles expressing TrkA-IR (~50 %) increased in L1 and L6 DRG. The percentage of bladder afferent cell profiles expressing TrkB-IR (~45 %) in L1, L2, L6, and S1 DRG also increased compared with control cell profiles [125]. The increase in TrkA-IR in bladder afferent cells occurred 8 h after CYP treatment and was maintained in L1 DRG with chronic (10 days) CYP-induced cystitis. However, the increase in bladder afferent cells expressing TrkB-IR only occurred at the most acute time point examined (8 h). TrkA-IR and TrkB-IR cell profiles also demonstrated phosphorylated Trk-IR with acute and/or chronic CYP-induced cystitis [125].

Spinal Cord Injury (SCI)

After SCI, a significant increase in the number of TrkB-immunoreactive cells was also detected in the L6–S1 DRG and in the L1–L2 DRG but not in the L4–L5 DRG compared with control rats [127]. After SCI, the percentage of FB-labeled cells expressing TrkA- or TrkB-IR in L1 and L6 DRG significantly increased compared with control DRG. After SCI, the percentage of TrkA-immunoreactive cells expressing phosphorylated (p)-Trk-IR significantly increased (1.5- to 2.3-fold increase) in the L1, L6, and S1 DRG. The percentage of TrkB-immunoreactive cells expressing p-Trk-IR after SCI also increased (1.3-fold increase) in the L1 and L6 DRG [127]. These results demonstrate that (1) TrkA- and TrkB-IR is increased in bladder afferent cells after SCI and (2) TrkA and TrkB receptors are phosphorylated in DRG after SCI. Neuroplasticity of LUT reflexes after SCI may be mediated by both NGF and brain-derived neurotrophic factor in target tissues [127].

NGF and PACAP Interactions

Recent reports have demonstrated reciprocal regulatory interactions between NGF and PACAP in rat pheochromocytoma (PC)12 cells and in DRG cells. Recent studies [128] have implicated NGF as a positive regulator of PACAP expression in nociceptive DRG cells. In rat PC12 cells, both NGF and PACAP can induce PC differentiation into a neuronal phenotype [129]. Upon PC12 transfection of a PACAP promoter-luciferase construct, exogenously applied PACAP and NGF, added either alone or in combination, upregulated PACAP gene expression [130, 131]. In addition, the neurotrophins can also facilitate expression of the PACAP-selective PAC1 receptor. NGF upregulated the PAC1 receptor promoter in PC12 cells; both NGF and BDNF induced PAC1 receptor promoter activity and mRNA expression in cerebellar granule cells [132].

Conversely, PACAP has also been shown to upregulate TrkA and TrkB receptor expression and/or phosphorylation in PC12 cells and hippocampal neurons, respectively in a Src-dependent manner [133]. Studies with sympathetic neuroblasts also demonstrated that PACAP can augment TrkA and TrkC expression in the neuronal differentiation process [134]. The ability for the PACAP and NGF signaling pathways to demonstrate reciprocal regulatory processes may be a primary example of an important feed-forward mechanism to amplify a trophic survival or differentiation response during neuronal development or regeneration. In bladder inflammation or other pathophysiological events, the same feed-forward mechanism may present complications and exacerbate dysfunction.

Transgenic Mouse Model with Chronic Urothelial Overexpression of NGF (NGF-OE)

Our laboratory has characterized a transgenic mouse model of urothelium-specific, NGF-OE that represents a novel approach to exploring the role of NGF in urinary bladder inflammation and sensory function [135]. Functionally, NGF-OE mice exhibit urinary bladder hyperreflexia with frequent urination and the presence of non-voiding bladder contractions as well as referred somatic pelvic hypersensitivity [135]. No changes in the electrical properties of the MPG neurons of NGF-OE mice were detected using intracellular recording, suggesting that the urinary bladder phenotype in NGF-OE mice is not influenced by changes in the efferent limb of the micturition reflex. NGF-OE mice may represent a useful animal model of BPS/IC because the changes observed in the urinary bladders of these mice are consistent with certain changes observed in this syndrome. Pleiotropic changes, subsequent to NGF-OE, including changes in the expression of growth factors, neuroactive compounds, and ion channels (e.g., transient receptor potential (TRP) channels) [136, 137] can also directly modulate pain and bladder/visceral sensory function and could contribute to altered urinary bladder function in NGF-OE mice [11, 137–139].

Recent studies also demonstrate changes in PACAP/VIP and receptor expression in micturition pathways in NGF-OE mice [140]. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-IR in urothelium of NGF-OE mice whereas PACAP transcript and PACAP-IR were decreased in urothelium of NGF-OE mice [140]. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice [140]. VIP transcript expression and VIP-IR was not altered in urinary bladder of NGF-OE mice [140]. Changes in PACAP, VIP and associated receptors transcripts and peptide expression in micturition pathways resemble some, but not all, changes observed after induction of urinary bladder inflammation known to involve NGF production.

Contributions of PACAP/Receptor Signaling to Increased Voiding Frequency and Somatic Sensitivity in NGF-OE

Given the presence of PAC1-IR fibers, the expression of PAC1 receptor expression in bladder tissues, and the abilities of PACAP to facilitate detrusor contractility, whether PACAP/receptor signaling contributes to bladder hyperreflexia and somatic sensitivity was recently evaluated [141]. Intravesical administration of PACAP6-38 (300 nM) significantly increased bladder capacity (2.0-fold), intercontraction interval and void volume in NGF-OE mice. Intravesical instillation of PACAP6-38 also decreased filling and peak micturition pressure in NGF-OE mice [141]. PACAP6-38 had no effects on WT mice. Intravesical administration of PACAP6-38 (300 nM) significantly reduced pelvic sensitivity in NGF-OE mice but was without effect in WT mice. PACAP/receptor signaling contributes to the increased voiding frequency and pelvic sensitivity observed in NGF-OE mice [141].

We have extended these studies to address the contribution of target-derived NGF in combination with CYP-induced cystitis to determine whether additional changes in neuropeptides/receptors are observed in micturition reflex pathways due to the presence of additional inflammatory mediators in the urinary bladder [142]. Quantitative polymerase chain reaction (PCR) was used to determine PACAP/ VIP, SP, galanin, and receptor transcript expression in the urinary bladder (urothelium, detrusor) in NGF-OE mice and wild type (WT) mice with CYP-induced cystitis (4 h, 48 h, and chronic) [142]. With CYP-induced cystitis (4 h), WT and NGF-OE mice exhibited similar changes in galanin transcript expression in the urothelium (30-fold increase) and detrusor (threefold increase). In contrast, PACAP, VIP, and SP transcripts exhibited differential changes in WT and NGF-OE with CYP-induced cystitis. PAC1, VPAC1, and VPAC2 transcript expression also exhibited differential responses in NGF-OE mice that were tissue (urothelium vs. detrusor) and CYP-induced cystitis duration-dependent [142]. Using conscious cystometry, NGF-OE mice treated with CYP exhibited significant increases in voiding frequency above that observed in control NGF-OE mice [142]. These studies are consistent with target-derived NGF and other inflammatory mediators affecting neurochemical plasticity and the reflex function of micturition pathways.

We now have determined whether additional changes in neuropeptides/receptors and growth factor/receptors are observed in the urinary bladder (urothelium, detrusor) and lumbosacral dorsal root ganglia (DRG) involved in micturition reflexes in NGF-OE mice with CYP-induced cystitis (Girard and Vizzard, unpublished observations). Quantitative PCR was used to determine NGF, BDNF, VEGF, and receptors (TrkA, TrkB, p75NTR) and PACAP/VIP and receptors (PAC1, VPAC1, VPAC2) transcripts expression in tissues from NGF-OE and wild type (WT) mice with CYP-induced cystitis (4 h, 48 h, and chronic). As expected in urothelium of control NGF-OE mice, NGF mRNA was significantly increased. Urothelial expression of NGF mRNA in NGF-OE mice treated with CYP (4 h 48 h, and chronic) was not further increased but maintained with all durations of CYP treatment evaluated. In contrast, CYP-induced cystitis (4 h 48 h, and chronic) in NGF-OE mice demonstrated significant regulation in BDNF, VEGF mRNA, TrkA, TrkB, and P75NTR in urothelium and detrusor smooth muscle. Similarly, CYP-induced cystitis (4 h 48 h, and chronic) in NGF-OE mice resulted in significant differential changes in WT and NGF-OE in transcript expression for NGF, BDNF, and receptors (TrkA, TrkB, p75NTR) and PACAP/VIP and receptors (PAC1, VPAC1, VPAC2) in lumbosacral DRG that was also CYP-induced cystitis duration-dependent. These studies are consistent with target-derived NGF and other inflammatory mediators affecting neurochemical plasticity and contributing to reflex function of micturition pathways.

Perspectives and Significance

PACAP (Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in diverse systems including micturition reflex pathways including expression in both neural and non-neural (e.g., urothelium) components. PACAP and associated receptors exhibit neuroplastic changes in expression and function with neural injury, inflammation, and diseases of the LUT. Changes in the PACAP/receptor system in micturition pathways may underlie and/or contribute to LUT dysfunction including the symptoms of urinary urgency, increased voiding frequency, nocturia, urinary incontinence, detrusor dyssynergia, and/or pain. The PACAP/receptor system in micturition reflexes may represent a potential target for therapeutic intervention.