Bladder Dorsal Root Ganglion Neurons Research Articles

Electrophysiological properties of lumbosacral primary afferent neurons innervating urothelial and non-urothelial layers of mouse urinary bladder

Pelvic nerve (PN) bladder primary afferent neurons were retrogradely labeled by intraparenchymal (IPar) microinjection of fluorescent tracer or intravesical (IVes) infusion of tracer into the bladder lumen. IPar and IVes techniques labeled two distinct populations of PN bladder neurons differentiated on the basis of dorsal root ganglion (DRG) soma labeling, dye distribution within the bladder, and intrinsic electrophysiological properties. IPar (Fast blue)- and IVes (DiI)-labeled neurons accounted for 91.5% (378.3±32.3) and 8% (33.0±26.0) of all labeled neurons, respectively (p<0.01), with only 2.0±1.2 neurons labeled by both techniques. When dyes were switched, IPar (DiI)- and IVes (Fast blue) labeled neurons accounted for 77.6% (103.0±25.8) and 22.4% (29.8±10.5), respectively (P<0.05), with 6.0±1.5 double-labeled neurons. Following IPar labeling, DiI was distributed throughout non-urothelial layers of the bladder. In contrast, dye was contained within the urothelium and occasionally the submucosa after IVes labeling. Electrophysiological properties of DiI-labeled IPar and IVes DRG neurons were characterized by whole-mount, in situ patch-clamp recordings. IPar- and IVes-labeled neurons differed significantly with respect to rheobase, input resistance, membrane capacitance, amplitude of inactivating and sustained K+ currents, and rebound action potential firing, suggesting that the IVes population is more excitable. This study is the first to demonstrate that IVes labeling is a minimally invasive approach for retrograde labeling of PN bladder afferent neurons, to selectively identify urothelial versus non-urothelial bladder DRG neurons, and to elucidate electrophysiological properties of urothelial and non-urothelial afferents in an intact DRG soma preparation.

Pirt reduces bladder overactivity by inhibiting purinergic receptor P2X3.

Pirt is a transmembrane protein predominantly expressed in peripheral neurons. However, the physiological and pathological roles of Pirt in hollow viscus are largely unknown. Here we show that Pirt deficiency in mice causes bladder overactivity. The density of α,β-meATP-induced currents is significantly reinforced in Pirt-deficient dorsal root ganglion (DRG) neurons. Pirt and P2X3 receptor co-localize in bladder nerve fibres and heterologous Pirt expression significantly reduces P2X3-mediated currents. Pirt interacts with P2X3 through the N-terminal 14 amino-acid residues. TAT-conjugated Pirt(N14) peptide (Pirt(N14)) is sufficient to inhibit P2X3 activation in bladder DRG neurons and to alleviate bladder overactivity in Pirt(-/-) mice. Pirt expression is decreased in the bladder of cyclophosphamide (CYP)-treated mice, a commonly used model of bladder overactivity. Importantly, Pirt(N14) administration reduces the frequency of bladder voiding and restores the voided volume of CYP-treated mice. Therefore, our results demonstrate that Pirt is an endogenous regulator of P2X3 in bladder function.

VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity

BackgroundThis work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments.ResultsIn addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na+ channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation.ConclusionsFor the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.

Expression of Vesicular Glutamate Transporters in Sensory and Autonomic Neurons Innervating the Mouse Bladder

VGLUTs, which are essential for loading glutamate into synaptic vesicles, are present in various neuronal systems. However, to our knowledge the expression of VGLUTs in neurons innervating the bladder has not yet been analyzed. We studied VGLUT1, VGLUT2 and VGLUT3 in mouse bladder neurons. We analyzed the expression of VGLUT1, VGLUT2 and calcitonin gene-related peptide by immunohistochemistry in the retrograde labeled primary afferent and autonomic neurons of BALB/c mice after injecting fast blue in the bladder wall. To study VGLUT3 we traced the bladder of transgenic mice, in which VGLUT3 is identified by enhanced green fluorescent protein detection. Most bladder dorsal root ganglion neurons expressed VGLUT2. A smaller percentage of neurons also expressed VGLUT1 or VGLUT3. Co-expression with calcitonin gene-related peptide was only observed for VGLUT2. Occasional VGLUT2 immunoreactive neurons were seen in the major pelvic ganglia. Abundant VGLUT2 immunoreactive nerves were detected in the bladder dome and trigone, and the urethra. VGLUT1 immunoreactive nerves were discretely present. We present what are to our knowledge novel data on VGLUT expression in sensory and autonomic neurons innervating the mouse bladder. The frequent association of VGLUT2 and calcitonin gene-related peptide in sensory neurons suggests interactions between glutamatergic and peptidergic neurotransmissions, potentially influencing commonly perceived sensations in the bladder, such as discomfort and pain.

Dorsal root ganglion neurons innervating pelvic organs in the mouse express tyrosine hydroxylase

Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons.

Hyperexcitability of convergent colon and bladder dorsal root ganglion neurons after colonic inflammation: mechanism for pelvic organ cross‐talk

Clinical studies reveal concomitant occurrence of several gastrointestinal and urologic disorders, including irritable bowel syndrome and interstitial cystitis. The purpose of this study was to determine the mechanisms underlying cross-organ sensitization at the level of dorsal root ganglion (DRG) after acute and subsided gastrointestinal inflammation. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and Fast Blue were injected into the distal colon and urinary bladder of male rats, respectively. Convergent DRG neurons were found in L1-L3 and L6-S2 ganglia with an average distribution of 14% +/- 2%. The resting membrane potential (RMP) of cells isolated from upper lumbar (UL) ganglia was -59.8 +/- 2.7 mV, whereas lumbosacral (LS) neurons were more depolarized (RMP = -49.4 +/- 2.1 mV, P < or = 0.05) under control conditions. Acute trinitrobenzene sulfonic acid (TNBS) colitis (3 days) decreased voltage and current thresholds for action potential firing in LS but not UL convergent capsaicin-sensitive neurons. This effect persisted for 30 days in the absence of overt colonic inflammation. The current threshold for action potential (AP) firing in UL cells was also decreased from 165.0 +/- 24.5 pA (control) to 85.0 +/- 19.1 pA at 30 days (P < or = 0.05), indicating increased excitability. The presence of a subpopulation of colon-bladder convergent DRG neurons and their persistent hyperexcitability after colonic inflammation provides a basis for pelvic organ cross-sensitization.