Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 μM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly ( P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly ( P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly ( P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.

Changes in urinary bladder function and somatic sensation may be mediated, in part, by inflammatory changes in the urinary bladder including the expression of chemokines. Male and female C57BL/6 mice were treated with cyclophosphamide (CYP; 75 mg/kg, 200 mg/kg, i.p.) to induce bladder inflammation (4 h, 48 h, chronic). We characterized the expression of CXC chemokines (CXCL9, CXCL10 and CXCL11) in the urinary bladder and determined the effects of blockade of their common receptor, CXCR3, at the level urinary bladder on bladder function and somatic (hindpaw and pelvic) sensation. qRT-PCR and Enzyme-Linked Immunoassays (ELISAs) were used to determine mRNA and protein expression of CXCL9, CXCL10 and CXCL11 in urothelium and detrusor. In urothelium of female mice treated with CYP, CXCL9 and CXCL10 mRNA significantly (p ≤ 0.01) increased with CYP treatment whereas CXC mRNA expression in the detrusor exhibited both increases and decreases in expression with CYP treatment. CXC mRNA expression urothelium and detrusor of male mice was more variable with both significant (p ≤ 0.01) increases and decreases in expression depending on the specific CXC chemokine and CYP treatment. CXCL9 and CXCL10 protein expression was significantly (p ≤ 0.01) increased in the urinary bladder with 4 h CYP treatment in female mice whereas CXC protein expression in the urinary bladder of male mice did not exhibit an overall change in expression. CXCR3 blockade with intravesical instillation of AMG487 (5 mg/kg) significantly (p ≤ 0.01) increased bladder capacity, reduced voiding frequency and reduced non-voiding contractions in female mice treated with CYP (4 h, 48 h). CXCR3 blockade also reduced (p ≤ 0.01) hindpaw and pelvic sensitivity in female mice treated with CYP (4 h, 48 h). CXC chemokines may be novel targets for treating urinary bladder dysfunction and somatic sensitization resulting from urinary bladder inflammation.

Complex organization of CNS and PNS pathways is necessary for the coordinated and reciprocal functions of the urinary bladder, urethra and urethral sphincters. Injury, inflammation, psychogenic stress or diseases that affect these nerve pathways and target organs can produce lower urinary tract (LUT) dysfunction. Numerous neuropeptide/receptor systems are expressed in the neural pathways of the LUT and non-neural components of the LUT (e.g., urothelium) also express peptides. One such neuropeptide receptor system, pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the LUT. Mice with a genetic deletion of PACAP exhibit bladder dysfunction and altered somatic sensation. PACAP and associated receptors are expressed in the LUT and exhibit neuroplastic changes with neural injury, inflammation, and diseases of the LUT as well as psychogenic stress. Blockade of the PACAP/PAC1 receptor system reduces voiding frequency in preclinical animal models and transgenic mouse models that mirror some clinical symptoms of bladder dysfunction. A change in the balance of the expression and resulting function of the PACAP/receptor system in CNS and PNS bladder reflex pathways may underlie LUT dysfunction including symptoms of urinary urgency, increased voiding frequency, and visceral pain. The PACAP/receptor system in micturition pathways may represent a potential target for therapeutic intervention to reduce LUT dysfunction.

High expression of VEGF is associated with immature angiogenesis within the urinary bladder wall and bladder afferent nerve sensitization, leading to visceral hyperalgesia and pelvic pain. Research suggests a shift in VEGF alternative splice variant (VEGF-Axxxa and VEGF-Axxxb) expression with several pathologies (e.g., neuropathic pain and inflammation) as well as differing effects on pain. Translational studies have also demonstrated increased total VEGF expression in the bladders of women with interstitial cystitis/bladder pain syndrome. In the present study, we quantified VEGF alternative splice variant expression in lower urinary tract tissues under control conditions and with cyclophosphamide (CYP)-induced cystitis. Using conscious cystometry and intravesical instillation of a potent and selective VEGF receptor 2 (VEGFR2) tyrosine kinase inhibitor (Ki-8751, 1 mg/kg) in Wistar rats (male and female) with acute and chronic CYP-induced cystitis and control (no CYP) rats, we further determined the functional effects of VEGFR2 blockade on bladder function. With VEGFR2 blockade, bladder capacity increased ( P ≤ 0.01) in male and female control rats as well as in male and female rats with acute ( P ≤ 0.05) or chronic ( P ≤ 0.01 or P ≤ 0.05, respectively) CYP-induced cystitis. Void volume also increased in female control rats ( P ≤ 0.01) and female rats with acute ( P ≤ 0.05) or chronic ( P ≤ 0.05) CYP-induced cystitis as well as in male control rats ( P ≤ 0.05) and male rats with chronic CYP-induced cystitis ( P ≤ 0.01). These data suggest that VEGF may be a biomarker for interstitial cystitis/bladder pain syndrome and that targeting VEGF/VEGFR2 signaling may be an effective treatment.

We recently reported the radiosynthesis and in vitro evaluation of [18F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([18F]FECIMBI-36) or ([18F]1), an agonist radioligand for 5HT2A/2C receptors in postmortem samples of human brain. Herein we describe the in vivo evaluation of [18F]FECIMBI-36 in vervet /African green monkeys by PET imaging. PET images show that [18F]FECIMBI-36 penetrates the blood-brain barrier and a low retention of radioactivity is observed in monkey brain. Although the time activity curves indicate a somehow heterogeneous distribution of the radioligand in the brain, the low level of [18F]FECIMBI-36 in brain may limit the use of this tracer for quantification of 5-HT2A/2C receptors by PET.

Neural injury, inflammation, or diseases commonly and adversely affect micturition reflex function that is organized by neural circuits in the CNS and PNS. One neuropeptide receptor system, pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the lower urinary tract. PACAP and associated receptors are expressed in the LUT and exhibit changes in expression, distribution and function in preclinical animal models of bladder pain syndrome (BPS)/interstitial cystitis (IC), a chronic, visceral pain syndrome characterized by pain, and LUT dysfunction. Blockade of the PACAP/PAC1 receptor system reduces voiding frequency and somatic (e.g., hindpaw, pelvic) sensitivity in preclinical animal models and a transgenic mouse model that mirrors some clinical symptoms of BPS/IC. The PACAP/receptor system in micturition pathways may represent a potential target for therapeutic intervention to reduce LUT dysfunction following urinary bladder inflammation.

25Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in 26 neurons it regulates synaptic transmission, which is thought to be a major mechanism through which 27 altered mTOR signaling leads to neurological disease. Although previous studies have delineated 28 postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, 29 the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR's role in 30 synaptic transmission may be complex-specific. To better understand each complex's role in synaptic 31 transmission, we genetically inactivated mTORC1 or mTORC2 in cultured mouse glutamatergic 32 hippocampal neurons. Inactivation of either complex reduced neuron growth and evoked EPSCs, 33 however, mTORC1 exerted its effects on eEPSCs at the postsynapse and mTORC2 at the presynapse. 34 Furthermore, inactivation of each complex altered specific modes of synaptic vesicle release, suggesting 35 that mTORC1 and mTORC2 differentially modulate postsynaptic responsiveness and presynaptic release 36 to optimize glutamatergic synaptic transmission. 37 38 39 40 41 42 43 44 45 103 Results 104 Inactivation of mTORC1 or mTORC2 in neurons, via Raptor or Rictor deletion, causes distinct effects on 105 downstream mTOR signaling. 106To investigate the effects of mTORC1 or mTORC2 inactivation on neurons, we cultured primary 107 hippocampal neurons isolated from P0-P1 Raptor flox/flox or Rictor flox/flox mice on astrocytes isolated from 108 P0-P1 wild-type mice. At the time of plating, we transduced the neurons with adeno-associated viruses 109 (AAVs) expressing either an mCherry-Cre fusion protein or mCherry alone, both driven by the hSyn 110 promoter, to generate knockout (Raptor-KO or Rictor-KO) or control (Raptor-Con or Rictor-Con) 111 neurons, respectively. To assess the efficiency of gene deletion in Raptor-KO and Rictor-KO neurons, we 112 performed quantitative immunofluorescence analysis for phospho-S6 (S240/244) (pS6), an indicator of 113 mTORC1 activity, and phospho-Akt (S473) (pAkt), an indicator of mTORC2 activity. In Raptor-KO neurons, 114 the mean fluorescence intensity of the pS6 signal was decreased relative to Raptor-Con neurons (Con: 1151.00 ± 0.05, Rap-KO: 0.25 ± 0.03, p < 0.001; Figure 1A1-3), verifying a reduction in mTORC1 activity. The 116 pAkt signal was, however, increased in Raptor-KO neurons relative to Raptor-Con neurons (Con: 1.00 ± 117