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

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

Expression of Vesicular Glutamate Transporters in Sensory and Autonomic Neurons Innervating the Mouse Bladder Pablo R. Brumovsky,* Rebecca P. Seal, Ke...

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Expression of Vesicular Glutamate Transporters in Sensory and Autonomic Neurons Innervating the Mouse Bladder Pablo R. Brumovsky,* Rebecca P. Seal, Kerstin H. Lundgren, Kim B. Seroogy, Masahiko Watanabe and G. F. Gebhart From the Pittsburgh Center for Pain Research, Department of Anesthesiology, University of Pittsburgh (PRB, RPS, GFG), Pittsburgh, Pennsylvania, Consejo Nacional de Investigaciones Científicas y Técnicas and School of Biomedical Sciences, Austral University, Buenos Aires (PRB), Argentina, Department of Neurology, University of Cincinnati (KHL, KBS), Cincinnati, Ohio, and Department of Anatomy, Hokkaido University School of Medicine (MW), Sapporo, Japan

Abbreviations and Acronyms CGRP ⫽ calcitonin gene-related peptide DRG ⫽ dorsal root ganglion eGFP ⫽ enhanced green fluorescent protein IR ⫽ immunoreactive LSC ⫽ lumbar sympathetic chain MPG ⫽ major pelvic ganglion TH ⫽ tyrosine hydroxylase TRP ⫽ transient receptor potential cation channel TRPV1 ⫽ TRP, subfamily V, member 1 VGLUT ⫽ vesicular glutamate transporter Accepted for publication November 6, 2012. Study received approval from the University of Pittsburgh and University of California-San Francisco institutional animal care use committees. Supported by National Institutes of Health Awards NS035790 and DK093525 (GFG), an IASP Early Career Research Award and an Austral University Grant (PRB). * Correspondence: School of Biomedical Sciences, Austral University, Av. Juan D. Perón 1500, B1629AHJ, Pilar, Buenos Aires, Argentina (telephone: 54 0230 448 2699; e-mail: [email protected]).

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Purpose: 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. Materials and Methods: 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. Results: 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. Conclusions: 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. Key Words: urinary bladder, vesicular glutamate transport proteins, pain, neurons, botulinum toxins THE bladder is profusely innervated by sympathetic, parasympathetic and primary afferent neurons.1,2 The main neurotransmitters involved in autonomic functions are noradrenaline and acetylcholine, which are produced by sympathetic and parasympathetic neurons, respectively. The presence of these neurotransmitters in neurons and nerve fibers is usually

indicated by the identification of associated molecules, such as TH, the norepinephrine transporter-1 (noradrenergic) and choline acetyltransferase or the vesicular acetylcholine transporter (acetylcholinergic). Like nonvisceral primary afferent neurons,3 visceral primary afferent neurons are classically described as glutamatergic and often capable of co-expressing

0022-5347/13/1896-2342/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION

http://dx.doi.org/10.1016/j.juro.2012.11.046 Vol. 189, 2342-2349, June 2013 RESEARCH, INC. Printed in U.S.A.

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Technical specifications of primary and secondary antibodies, and fluorophores Dilution Host species ⫹ target antigen: Rabbit anti-VGLUT1 Guinea pig anti-VGLUT2 Rabbit anti-EGFP Rabbit anti-CGRP Secondary antibodies/fluorophores: Horseradish peroxidase-donkey anti-guinea pig Horseradish peroxidase-donkey anti-rabbit Tetramethylrhodamine isothiocyanate-donkey anti-rabbit Fluorescein tyramide

1:4,000 (tyramide signal amplification) 1:8,000 (tyramide signal amplification) 1:4,000 (tyramide signal amplification) 1:8,000 (indirect fluorescence)/1:40,000 (tyramide signal amplification) 1:200 1:200 1:400 1:700

various molecules, such as neuropeptides, various types of receptors4 and even autonomic neuron markers (TH).5 The earliest morphological study of the glutamatergic nature of bladder DRG neurons was reported more than 12 years ago, when Keast and Stephensen observed immunohistochemically detectable glutamate in approximately 50% of rat bladder DRG neurons.6 For use in neurotransmission glutamate is taken up into synaptic vesicles by VGLUTs. Different types of VGLUTs were recently identified, resulting in the reliable immunohistochemical characterization of various subpopulations of glutamatergic neurons in the central and peripheral nervous systems.7 In the latter system VGLUTs were characterized in rodent nonvisceral8 and colorectal primary afferent neurons,9 and in some LSC neurons after peripheral nerve injury.10 To our knowledge no analysis of the presence of VGLUTs in DRG and autonomic neurons projecting to the bladder has been available to date. We present a comprehensive immunohistochemical study of the expression of the 3 known VGLUTs in DRG, LSC and MPG neurons innervating the mouse bladder.

MATERIALS AND METHODS Male BALB/c mice (Taconic, Germantown, New York) at age 7 to 8 weeks and 129S6/SvEvTac-C57Bl/6 mice11 at age 6 weeks were used in all experiments. All research protocols adhered to the Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh and University of California-San Francisco institutional animal care use committees. None of the currently available commercial antibodies against VGLUT3 efficiently label DRG neurons. Therefore, we also used a transgenic mouse expressing eGFP under the control of VGLUT3 regulatory sequences that reliably identify VGLUT3 expressing neurons in these mice.11 The bladders of 5 BALB/c male mice and 3 VGLUT3eGFP male mice at age 6 weeks were injected with the

Reference/Source Kawamura Y et al: J Neurosci 2006; 26: 2991 Miyazaki T et al: Eur J Neurosci 2003; 17: 2563 ⫹ Brumovsky et al8–10 Molecular Probes® (catalogue No. A-11122) Sigma® (product A8198)

Jackson ImmunoResearch®, West Grove, PA (code No. 713-035-003) Jackson ImmunoResearch (code No. 711-035-152) Jackson ImmunoResearch (code No. 711-025-152) PerkinElmer® (product No. NEL741)

fluorescent retrograde neuronal tracer fast blue (2% in saline) (EMS-Chemie, Gross-Umstadt, Germany) according to previously reported surgical procedures.9 At 12 days after fast blue injection the mice were deeply anesthetized using sodium pentobarbital (60 mg/kg intraperitoneally) (Ovation Pharmaceuticals, Deerfield, Illinois) and perfused via the ascending aorta with previously described fixative mixtures.9 Three naïve BALB/c mice were similarly anesthetized and perfused. Thoracolumbar (T8-L1) and lumbosacral (L6-S2) DRGs, and the MPG, LSC and bladder were dissected out and processed according to previously described immunohistochemistry protocols.8,9 The table lists the antibodies used. Further details on these antibodies were published previously.9 We performed single or double staining experiments using TSA™ Plus and/or indirect immunofluorescence techniques.9 Briefly, sections were incubated with 1 and 2 antibodies for single and double staining, respectively, and processed first using the tyramide signal amplification technique. Additional tyramide signal amplification or indirect immunofluorescence protocols were used in double staining experiments. Nonspecific staining by secondary antibody was tested in a few sections by omitting the primary antibody. CGRP, VGLUTs and eGFP antiserum were previously described in detail.8,9 Immunofluorescence sections were examined using an Eclipse E600 microscope (Nikon, Tokyo, Japan) provided with a Retiga™ 2000 R Fast CCD camera using IPLab (Scanalytics, Vancouver, British Columbia, Canada). Colocalization was analyzed with a FluoView FV 1000 confocal microscope (Olympus®). Image resolution, brightness and contrast were optimized using Adobe® Photoshop® CS3. Because confocal imaging of fast blue was not possible due to the lack of appropriate filters, some MPG images were composed by merging separate optical (fast blue) and confocal (other markers of interest) photomicrographs. Retrograde traced neuron profiles were quantified in every fifth section of thoracolumbar and lumbosacral DRGs (5 to 8 sections per ganglion). The percent of VGLUT expressing bladder neuron profiles was determined by comparing the total number of fast blue positive neurons to those expressing each marker. Cell body diameters of a representative sample of each type of quantified

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neuron profile (more than 7 and up to 48 neurons per type) were measured using ImageJ (http://rsbweb.nih.gov/ij/). These data were used to correct raw counts based on the Abercrombie correction factor.12 Only neuron profiles showing immunostaining intensity higher than 2 orders of magnitude were counted. Data are presented as the mean ⫾ SEM. They were statistically analyzed using the Student t test for independent samples with p ⬍0.05 considered significant.

RESULTS Retrograde tracing from the bladder revealed a discrete number of fast blue positive neuron profiles of different sizes per section in thoracolumbar and lumbosacral DRGs, which were easily differentiated from fast blue negative neuron profiles (figs. 1 and 2). All VGLUTs were expressed in mouse DRG neurons projecting to the bladder, although in different proportions (fig. 1). An average of 32.4% ⫾ 3.6%, 94.1% ⫾ 3.0% and 18.1% ⫾ 1.3% of bladder DRG neuron profiles expressed VGLUT1, VGLUT2 and eGFP-VGLUT3, respectively. Further analysis revealed that VGLUT expression differed between thoracolumbar and lumbosacral DRGs. VGLUT1 was present in a greater proportion of thoracolumbar than lumbosacral bladder DRG neuron profiles (mean 38.9% ⫾ 4.2% vs 25.9% ⫾ 3.6%, p ⬍0.05, fig. 1, A to C). Most VGLUT1-IR neuron profiles appeared to be medium to large (fig. 1, A to C). VGLUT2 was detected in similar high proportions in thoracolumbar and lumbosacral bladder DRG neuron profiles of all sizes (mean 93.8% ⫾ 2.0% and 94.3% ⫾ 4.1%, respectively, fig. 1, D to I). Occasional bladder DRG neurons lacking VGLUT2 were also found (fig. 1, G to I). eGFP-VGLUT3 was expressed in a greater proportion of thoracolumbar than lumbosacral small bladder DRG neuron profiles (mean 27.9% ⫾ 2.0% vs 8.3% ⫾ 0.6%, p ⬍0.001, fig. 1, J and L). Finally, in addition to bladder neurons, abundant VGLUT2 or eGFP-VGLUT3-IR and several VGLUT1-IR fast blue negative neurons were detected in all DRGs (figs. 1 and 2). Colocalization analysis showed that only VGLUT2 was co-expressed with CGRP in bladder DRG neuron profiles (fig. 2). Thus, while no VGLUT1 bladder DRG neuron profiles co-expressed CGRP (fig. 2, A to D), a mean of 54.7% ⫾ 3.4% of thoracolumbar and 52.2% ⫾ 9.9% of lumbosacral bladder DRG neuron profiles co-expressed VGLUT2 and CGRP (fig. 2, E to H). We also detected fast blue negative VGLUT2-IR neuron profiles that co-expressed CGRP as well as CGRP only bladder DRG neurons (fig. 2, A to D, F and I to L). As observed for VGLUT1, virtually no eGFP-VGLUT3 bladder DRG neuron profiles co-expressed CGRP (fig. 2, I to L). This lack of co-expression with CGRP was also noted

Figure 1. VGLUT expression in DRGs innervating bladder. Optical immunofluorescence photomicrographs show sections of S1 (A to C), L6 (D to F), T10 (G to I) and T11 (J to L) DRGs incubated with VGLUT1 (B), VGLUT2 (E and H) and eGFPVGLUT3 (K) antiserum. Merged images (C, F, I and L) show retrograde labeled bladder DRG neurons containing fast blue (A, D, G and J) (purple areas). Green areas represent VGLUT1, VGLUT2 or eGFP-VGLUT3-IR neurons. White areas represent VGLUT and fast blue colocalization. Double arrowheads indicate VGLUT1 (A to C), VGLUT2 (D to I) and eGFP-VGLUT3 (J to L) IR bladder neuron profiles. Arrows indicate bladder neuron profiles lacking VGLUT1, (A), VGLUT2 (G) and eGFP-VGLUT3 (J)-like immunoreactivity. Arrowheads indicate nontraced neuron profiles expressing VGLUT1 (B), VGLUT2 (E) and eGFP-VGLUT3 (K). Scale bars represent 50 ␮m (G to I, and A to F and J to L).

for fast blue negative VGLUT1 and eGFP VGLUT3-IR neuron profiles (fig. 2, A to D and I to L). We also observed retrograde labeling of LSC and MPG bladder neurons (fig. 3, A, D, G, J, M and P). In the LSC no fast blue positive neurons expressed VGLUT1 or VGLUT2 (fig. 3, A to F). Nevertheless, some VGLUT2-IR fibers and what appeared to be VGLUT2-IR synaptic varicosities were detected in the LSC (fig. 3, D to F). Similarly, fast blue positive neurons in the MPG lacked VGLUT1-like immunoreactivity (fig. 3, G to I), which was only associated with a few varicose profiles (fig. 3, H and I). In contrast, occasional VGLUT2-IR bladder MPG neurons were found (fig. 3, J and L), along with many VGLUT2-IR fibers and varicosities (fig. 3, K, L, N, O, Q and R). Some varicosities with a basket-like appearance surrounded nonbladder MPG neurons (fig. 3, P and R). In the bladder VGLUT1-like immunoreactivity was commonly observed in the muscular and to a lesser extent in the submucosal layers of the bladder

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Figure 2. VGLUT-CGRP co-expression patterns in DRGs innervating bladder. Optical immunofluorescence photomicrographs show sections of T10 (A to D), L6 (E to H) and S2 (I to L) DRGs incubated with VGLUT1 (B), VGLUT2 (F) and eGFP-VGLUT3 (J) antiserum. In merged images (D, H and L) red areas represent retrograde labeled bladder DRG neurons containing fast blue (A, E and I). Green areas represent VGLUT1, VGLUT2 or eGFP-VGLUT3-IR neurons. Blue areas represent CGRP-IR neurons. White areas represent VGLUT plus fast blue plus CGRP colocalization. Orange areas indicate VGLUT plus fast blue. Purple areas indicate CGRP plus fast blue. Double arrowheads indicate VGLUT1 (A to D), VGLUT2 (E to H) and eGFP-VGLUT3 (I to L) IR bladder neuron profiles lacking CGRP-like immunoreactivity. Black double arrowheads indicate bladder neuron profiles expressing CGRP (A to D) and lacking VGLUT-like immunoreactivity (I to L). Double arrows indicate VGLUT2-IR bladder neuron profiles co-expressing CGRP (E to H). White arrowheads indicate nontraced VGLUT1 (B), VGLUT2 (F) and eGFP-VGLUT3 (J) IR DRG neuron profiles lacking CGRP-like immunoreactivity. Black arrowheads indicate nontraced VGLUT2-IR neuron profiles co-expressing CGRP (E to H). Scale bar represents 50 ␮m (L).

(fig. 4, A and B). In contrast, VGLUT2-IR nerves formed a profuse fiber network spanning all layers of the bladder wall throughout the dome and trigone, and the urethra (fig. 4, C to G). Colocalization analysis demonstrated that VGLUT1 and CGRP coexpression in bladder nerve fibers was virtually nonexistent (fig. 5, A to C). In contrast, VGLUT2 and CGRP were often colocalized in nerve fibers in the bladder (fig. 5, D to L).

DISCUSSION Although sensory neurons are often glutamatergic,6 information on the types of VGLUTs involved in the

synaptic vesicle uptake of glutamate in such neurons has only recently received attention.8 Evidence of VGLUT expression in visceral sensory neurons was first reported by Tong et al in ganglia innervating the rat stomach.13 Subsequently, VGLUT expression was confirmed in the peripheral projections of vagal and DRG neurons innervating the gut of the guinea pig, rat and mouse.9 More recently, we reported that more than 95% of mouse colorectal DRG neurons express VGLUT2 as well as VGLUT1 but the latter in a much lower proportion.9 Similarly, the current study shows that mouse bladder DRG neurons also predominantly express VGLUT2 and to a lesser extent VGLUT1 or VGLUT3.

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pression of various molecules in afferent neurons comprising the 2 innervation pathways.5 For example, 66% of mouse thoracolumbar bladder neurons express mRNA of TRP, subfamily A, member 1, while expression in lumbosacral bladder neurons is sparse at about 2.5%.14 In contrast, TH is expressed in threefold (colorectum) and fivefold (bladder) greater proportions in lumbosacral DRGs.5 The differences in VGLUT1 and VGLUT3 expression between thoracolumbar and lumbosacral bladder neurons that we report could imply differences in neuronal physiology. This was already reported in the mouse colorectum, in which higher TRPV1 expression in thoracolumbar than in lumbosacral DRGs corresponds to a stronger response to applied

Figure 3. VGLUT expression in LSCs and MPGs innervating bladder. Optical (A, D, G, J, M and P) and confocal (B, C, E, F, H, I, K, L, N, O, Q and R) immunofluorescence photomicrographs show LSC (A to F) and MPG (G to R) sections after incubation with VGLUT1 (B and H) and VGLUT2 (E, K, N and Q) antiserum. Merged images (C, F, I, L, O and R) show retrograde labeled bladder DRG neurons containing fast blue (purple areas) (A, D, G, J, M and P). Green areas represent VGLUT1 and VGLUT2-IR neurons. White areas represent VGLUT plus fast blue colocalization. Arrows indicate bladder neuron profiles in LSC (A and D) and MPG (G, J and P) lacking VGLUT1 and VGLUT2-like immunoreactivity, respectively. White arrowheads indicate VGLUT1 or VGLUT2-IR varicosities in LSC (E) and MPG (H, K and Q). Note higher magnification (I) of arrowhead (H). Black arrowheads indicate VGLUT2-IR fibers in LSC (E) and MPG (K and Q). VGLUT2-IR varicosities (arrowhead and arrow) surrounding bladder neuron profiles lacking transporter were detected (F and inset). Small double arrows indicate VGLUT2-IR bladder neuron profiles (J and L). Note higher magnification (M to O). White double arrowhead in panel shows VGLUT2-IR basket surrounding nontraced neuron profiles lacking transporter (Q and R, inset). Scale bars represent 50 (A to Q) and 10 (insets) ␮m.

Like the colorectum, the bladder is innervated by lumbar splanchnic and pelvic nerves with cell bodies in thoracolumbar and lumbosacral DRGs, respectively. We found that bladder sensory neurons expressing VGLUT 1 or VGLUT 3 , unlike those synthesizing VGLUT2, are more abundant in thoracolumbar than in lumbosacral DRGs. An increasing number of reports have revealed significant differences in the function and neurochemical ex-

Figure 4. Confocal immunofluorescence photomicrographs show transverse (A to D) and sagittal (E to G) sections of bladder dome (A to D), trigone (E) and urethra (F and G) after incubation with VGLUT1 (A and B) and VGLUT2 (C to G) antiserum. White double arrowheads indicate VGLUT1 (A and B) and VGLUT2 (C and D) IR nerve fibers in bladder muscular layer. White arrowheads indicate VGLUT1 (B) and VGLUT2 (C and D) IR nerve fibers in bladder submucosal layer. Arrows indicate VGLUT2-IR nerve fibers in bladder trigone (E). Black arrowheads (F and G) indicate VGLUT2-IR fibers penetrating urethra. White arrowheads indicate VGLUT2-IR nerve fibers innervating urethra (F and G). Scale bars represent 100 ␮m (A to D and E to G).

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Figure 5. Confocal immunofluorescence photomicrographs show transverse sections of bladder after co-incubation with VGLUT1 (A), VGLUT2 (D, G and J) and CGRP (B, E, H and K) antiserum. In merged images green areas represent VGLUT1 or VGLUT2-IR fibers (C, F, I and L). Purple areas represent CGRP-IR fibers. White areas represent VGLUT plus CGRP colocalization. Arrowheads indicate VGLUT1 (A) and VGLUT2 (D) IR nerve fibers lacking CGRP-like immunoreactivity in bladder dome. Arrows indicate CGRP-IR nerve fibers lacking VGLUT-like immunoreactivity in bladder dome muscular layer (B and H). Double arrows indicate VGLUT2 (G to I) and CGRP (J to L) IR nerve fibers in bladder mucosal (inset 1) and muscular (inset 2) layers (F) at higher magnification. Scale bars represent 50 ␮m (D to F) and 25 (A to C and G to K) ␮m.

capsaicin at colorectal thoracolumbar nerve terminals.15 We found that about half of the 94% of retrograde traced bladder DRG neurons expressing VGLUT2 co-expressed the peptidergic marker CGRP. These values correlate with previous studies in rats showing that up to 69% of DRG neurons innervating the bladder express CGRP.4 In the mouse colorectum more than 80% of all retrograde traced DRG neurons co-expressed VGLUT2 and CGRP,9 in accordance with CGRP expression in most DRG neurons

innervating the rodent colorectum.4 Accordingly, a large proportion of visceral glutamatergic sensory neurons may also synthesize and potentially co-release CGRP. In contrast, in DRGs innervating nonvisceral tissues only about 31% of the approximately 65% VGLUT2-IR neurons colocalize CGRP,8 suggesting potentially variable levels of co-release of glutamate and CGRP by DRG neurons that innervate different tissues. VGLUT1 and VGLUT3-IR bladder neurons did not co-express CGRP. However, since virtually all

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neurons innervating this organ synthesized VGLUT2, it is highly likely that VGLUT1 and/or VGLUT3 is co-expressed with VGLUT2 in its nonpeptidergic subpopulation, as described. Co-expression of more than 1 type of VGLUT was first reported for some neurons in the central nervous system16 and later confirmed for VGLUT1 and VGLUT2 in nonvisceral primary afferent neurons and their projections.8 Our study in the mouse colorectum9 and our current bladder series further support the supplementary contribution of different VGLUTs to glutamatergic physiology in sensory neurons. TRPV1 is a nonselective cation channel activated by pH, heat and capsaicin that is strongly implicated in bladder nociception.17 TRPV1 is expressed in many peptidergic bladder DRG neurons in the rat18 and mouse.14,15,18 Considering the prominent VGLUT2 expression in peptidergic bladder DRG neurons, VGLUT2 undoubtedly coexists with TRPV1. In support of this notion, VGLUT2 co-expression with TRPV1 was previously noted in nerve fibers terminating in the mouse rectum.19 Interestingly, TRPV1 and glutamate receptors also colocalize in nonvisceral small diameter primary afferent neurons and the induction of c-Fos expression in the spinal dorsal horn induced by subcutaneous capsaicin injection has been prevented by concomitant subcutaneous administration of ionotropic or metabotropic glutamatergic receptor antagonists.20 Peripheral release of glutamate, likely from primary afferent nerve terminals, was noted in the hind paw skin of rats exposed to intraplantar injection of the irritant formalin.21 Moreover, local injection of glutamate induces nociceptive behavior in rats22 and humans.23 Thus, we speculate that TRPV1-glutamate receptor interaction at central and peripheral nerve terminals, potentially driven by glutamate released from primary afferent neurons, could have a role in the physiopathology of bladder sensation and pain. As suggested, bladder nociception could be influenced by the peripheral release and action of glutamate. In recent years botulinum toxin A has been used to treat bladder pain associated with interstitial cystitis. Although botulinum toxin A was originally described as a potent inhibitor of acetylcholine release in the neuromuscular junction,24 it is increasingly accepted that botulinum toxin A also blocks transmitter release at nonacetylcholinergic synapses. Thus, it was hypothesized that botulinum toxin A desensitizes peripheral afferent nerves by inhibiting vesicular release of adenosine triphosphate and the release of peptides such as CGRP and substance P. Reduction of the axonal expression of TRPV1 from urothelial and suburothelial nerve endings was also proposed.25

In humans synaptic vesicle protein 2, a high affinity receptor for botulinum toxin A, and synaptosomal-associated protein 25, one of the soluble NSFattachment protein receptors involved in vesicle fusion before neurotransmitter release that is essential for the blocking action of botulinum toxin A, was found in at least half of bladder peptidergic nerve fibers.26 In rodents most nonvisceral,8 colorectal9 and bladder (current study) DRG neurons/nerves that express VGLUT2 and often synthesize peptides most likely have the soluble NSF-attachment protein receptor machinery for synaptic neurotransmitter release. Therefore, we speculate that in these neurons/nerves botulinum toxin A could also affect glutamatergic synaptic vesicle docking, limiting glutamate release and, thus, contributing to the analgesic effect of the toxin. In fact, the benefits of botulinum toxin were successfully explored in animals with nonvisceral neuropathy.24 Finally, we detected a few MPG neurons projecting to the bladder that also expressed VGLUT2, while no retrograde traced neurons in the LSC showed positive immunostaining for any VGLUT. Thus, in normal conditions DRGs as well as the MPG may be a source of VGLUT2-IR fibers in the bladder. Interestingly, we recently reported that a subpopulation of LSC neurons up-regulates VGLUT2 upon injury to the pelvic nerve.10 In that study we did not trace the neurons. Thus, to our knowledge it remains to be established whether some LSC neurons that up-regulate VGLUT2 project to the bladder or colorectum. Likewise, we speculate that MPG neurons that do or do not project to visceral organs may up-regulate VGLUT2 after injury to their postganglionic axons.

CONCLUSIONS We present an immunohistochemical account of VGLUT expression in sensory and autonomic neurons innervating the mouse bladder. The finding of a preponderant expression of VGLUT2, as recently also noted in the mouse colorectum,9 suggests a relevant role for this transporter in glutamatergic neurotransmission in neurons innervating pelvic visceral organs, from which the principal conscious sensations that arise are discomfort and pain. Glutamate, which is contained in the central terminals of primary afferents, spinal interneurons and terminals of fibers that descend from the medulla oblongata,27 is well established as important for nociceptive transmission from pelvic organs.1,28 Thus, complex interaction was noted between spinal ionotropic (NMDA and AMPA) receptors in the spinal processing of nociceptive input from the irritated

EXPRESSION OF VESICULAR GLUTAMATE TRANSPORTERS IN NEURONS INNERVATING BLADDER

lower urinary tract.29 Likewise, a facilitatory role in bladder primary afferent processing was attributable to metabotropic glutamate receptor 5 in normal conditions and during lower urinary tract inflammation.28 However, the role of peripheral glutamate release in the bladder is unclear. Thus, the intravesical application of metabotropic glutamate receptor antagonists has no effect on bladder contractility and pelvic nerve afferent firing in normal rats.30 Studies in animal disease models are needed to es-

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tablish the potential involvement of peripheral glutamate/glutamatergic receptors in the bladder.

ACKNOWLEDGMENTS Tim McMurray provided technical assistance. Drs. Jun-Ho La and Kathryn Albers, Center for Pain Research, University of Pittsburgh, and Dr. Carly McCarthy, School of Biomedical Sciences, Austral University, provided assistance.

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