Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat

Neuroscience Letters, 147 (1992) 33-36

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© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 09084

Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat D a n i e l L. McNeill, Neil E. T r a u g h Jr., A t u l M. Vaidya, H u o n g T. H u a a n d R a y m o n d E. P a p k a Department of Anatomical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 (USA)

(Received 22 July 1992;Accepted 17 August 1992) Key words: Urinary bladder; NADPH; Diaphorase; Nitric oxide synthase; Major pelvic ganglion; Dorsal root ganglion; Inferior mesentericgan-

glion Nicotinamide adeninedinucleotidephosphate (NADPH)-diaphorasehistochemistrywas utilizedto localizenitric oxide synthase(NOS), and thus sites where nitric oxide (NO) can be synthesized,within peripheral nervous system perikarya and fibers. Recent studies suggest that NO relaxes vascular and non-vascular smooth muscle. In this study, the origin and distribution of NADPH-diaphorase perikarya and fibers in the rat urinary bladder wereexamined.Results suggestthat a smallnumber of NADPH-diaphorase-positiveperikaryaare present withinthe bladder wall and within adjacent small ganglia. In addition, NADPH-diaphorase-positivenerve fibers were observed in the adventitialand muscular layers, subjacent to the urothelium and as perivascularfibers. After injectionof the retrograde tracer fluorogold(FG) into the bladder wall, numerous FG-labeledperikarya in the major pelvic ganglia and the TI3-L2, L6 and S~ dorsal root ganglia were NADPH-diaphorase positive. However, none of the FG-labeled perikarya in the inferior mesentericganglia were NADPH-diaphorasepositive.The prevalenceof NADPH-diaphorase-positiveperikarya and fibers suggests that NO may serve a role in bladder function.

While numerous studies have examined the effects of cholinergic, noradrenergic, purinergic and peptidergic compounds on bladder contraction (for review see ref. 8), recent studies have implicated nitric oxide (NO) as an important transmitter/messenger in autonomic neurotransmission [ 2 4 , 13, 15, 18, 19]. To indirectly visualize NO, the histochemical stain for the enzyme nicotinamide adenine dinucleotide phosphate (reduced) (NADPH-diaphorase) is used. Hope et al. [12] demonstrated that neuronal NADPH-diaphorase is identical to nitric oxide synthase (NOS) and NOS is the enzyme responsible for synthesis o f NO. Functionally, Thornbury et al. [18] observed that neurogenic relaxation of the sheep bladder neck was mediated by NO. In anatomical studies, Crowe et al. [5] and Gabella [10] used a histochemical method to identify numerous NADPH-diaphorase-positive neuronal perikarya within the bladders of newborn and adult guineapigs, respectively. Moreover, the urinary bladder receives an ample postganglionic efferent innervation from

Correspondence: D.L. McNeill, Department of Anatomical Sciences, University of Oklahoma, P.O. Box 26901, Oklahoma City, OK 73190, USA. Fax: (1) (405) 271-3548.

perikarya located in the inferior mesenteric ganglion (IMG), paravertebral chain ganglia, major pelvic ganglia (MPG), small ganglia adjacent to the bladder and intrinsic perikarya [7, 8, 16, 17]. In addition, bladder afferent nerve fibers originate from perikarya located within the TIa-L2 and t 6 and $1 dorsal root ganglia (DRG) [14]. Thus, given the possible importance of N O to bladder function, the present study was undertaken to characterize the distribution of intrinsic NADPH-diaphorase-positive perikarya and nerve fibers in the rat bladder. In addition, the retrograde tracer fluorogold was utilized to examine the origin of bladder NADPH-diaphorase-positive nerve fibers. Five male Sprague-Dawley rats (150-200 g; Sasco, Omaha, NE) were maintained on a 12:12 h, light:dark photoperiod and provided food and water ad libitum. Two rats were deeply anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and perfused transcardially with 300 ml of 0.9% saline followed by 300 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.2 at room temperature. Following the perfusion, each bladder was transected at its junction with the external urethral sphincter, placed in fresh fixative for 12 h and then in 30% sucrose in PB for 24 h. The whole bladders were then serially sectioned (20/.tm) using a cryostat.

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Fig. 1. Cryostat sections of rat urinary bladder stained for NADPH-diaphorase reactivity (A,B). In A, a small ganglion adjacent to the detrusor muscle contains two NADPH-diaphorase-positive perikarya (large arrows) and two unstained perikarya surrounded by NADPH-diaphorasepositive varicosities (small arrows). In B, NADPH-diaphorase-positive nerve fibers (arrows) are apparent coursing within the muscle bundles. Photomicrographs C and D are from the same L2 DRG section. In C, a single FG-labeled bladder neuron (arrow) is evident which is also stained for NADPH-diaphorase reactivity (arrow in D). U, urothelium; L, lumen; M, detrusor muscle. A, 300x; B, 420x; C and D, 620×.

Sections were thawed onto gelatinized slides and allowed to air dry. Sites of N O synthesis in neuronal perikarya and fibers were demonstrated with the N A D P H - d i a p h o r a s e histochemical technique since neuronal N A D P H - d i a p h o r a s e is an N O synthase [12]. The slide-mounted sections of bladder were hydrated in 0.1 M PB, p H 7.4 for 10 min, then incubated in a solution containing 1.0 mg/ml flN A D P H , 0.25 mg/ml nitroblue tetrazolium, 0.3% Triton X-100 (all from Sigma Chemical Co., St. Louis, MO) in PB, p H 7.4 for 50 min at 37°C. The reaction was stopped by multiple washings in PB, the sections were coverslipped with glycerin:PB and viewed using an Olympus Vanox microscope. As a control for the N A D P H - d i a phorase reaction, additional cryostat sections were processed in media in which the substrate had been omitted. The retrograde fluorescent tracer fluorogold (FG) was utilized to determine the origin of bladder N A D P H - d i a phorase-positive nerve fibers. Three rats were anesthetized with sodium pentobarbital, the bladder exposed and a small puncture made through the adventitia on the

dorsal surface of the bladder near the base. Prior to the surgery, small pledgets of gelfoam (approximately 0.5 m m 2) were saturated with F G and allowed to air dry. A single pledget was subsequently placed through the puncture wound, thus lying adjacent to the detrusor muscle. The puncture wound was sealed with New-Skin (Medtech Labs, Cody, WY), the abdominal muscles and skin sutured in layers and the animals allowed to recover. Ten days post-surgery, each rat was reanesthetized, perfused transcardially as previously described and the M P G , I M G and the T~3-L2, L6 and Sj D R G were removed and placed in fresh fixative for 12 h, transferred to 30% sucrose in PB for 24 h then serially sectioned (20 pro) using a cryostat. Sections were thawed onto gelatinized slides and allowed to air dry. FG-labeled perikarya in alternate sections of the D R G and I M G were photographed using an Olympus microscope equipped with ultraviolet epi-illumination and their coordinates recorded. Due to the large number of FG-labeled perikarya in the MPG, five sections/ganglion were photographed and their coordinates recorded. Sub-

35 sequently, the slide-mounted sections were processed for NADPH-diaphorase reactivity as previously described. Using the coordinates, FG-labeled perikarya in each ganglion section were relocated and rephotographed using brightfield microscopy. By comparing the two photographs, the percentage of bladder FG-labeled perikarya reactive for NADPH-diaphorase in each ganglion was estimated. In the cryostat sections of urinary bladder, several individual NADPH-diaphorase-positive perikarya (approximately 24/bladder) were observed embedded within the detrusor muscle. In addition, two to four N A D P H diaphorase-positive perikarya were located within small ganglia subjacent to the bladder adventitia (Fig. 1A). O f particular interest was the presence o f NADPH-diaphorase-positive varicosities surrounding unreactive perikarya (Fig. 1A). Fine NADPH-diaphorase-positive nerve fibers were frequently observed in small nerves and as individual fibers within bundles of detrusor muscle fibers (Fig. I B). NADPH-diaphorase-positive nerve fibers were also distributed in the adventitial layer, subjacent to the urothelium and as perivascular plexi. In the I M G (n=3) of 78 FG-labeled bladder perikarya examined, none were NADPH-diaphorase-positive. In fact, no NADPH-diaphorase reactivity was observed in any of the I M G perikarya. However, of the 386 FGlabeled bladder perikarya in the M P G (n--3), 70 (18.1%) were NADPH-diaphorase-positive. O f the FG-labeled bladder afferent perikarya in the D R G (T13=38, LI=81, L2=57, L6=255, S1=30; n=3 for each level), 28.9% at Tl3 , 42.0% at L1, 54.4% at L 2, 38.0% at L 6 and 56.7% at $1 were NADPH-diaphorase positive (Fig. 1C,D). In control sections incubated in the absence of substrate, NADPH-diaphorase reactivity was not observed. The purpose of this study was to characterize the distribution and origin of NADPH-diaphorase-positive perikarya and fibers in the rat urinary bladder. Our observations indicate there is a small population of NADPH-diaphorase neuronal perikarya that are intrinsic to the detrusor muscle and located within small ganglia subjacent to the bladder adventitia. Individual NADPH-diaphorase-positive perikarya did not demonstrate a predilection for a particular region of the bladder, but were evenly distributed throughout the detrusor muscle. This distribution is similar to that observed in the mouse [11]. Of interest is the apparent species difference between the small number of NADPH-diaphorasepositive intrinsic perikarya we observed in the rat bladder (approximately 24/bladder) versus the relatively large number (2000-2500 perikarya/bladder) reported in the guinea-pig bladder [10]. NADPH-diaphorase-positive nerve fibers were evident throughout the bladder, but were generally more abundant near the bladder base.

Postganglionic sympathetic perikarya innervating the bladder have been reported in paravertebral chain ganglia and in the I M G [8]. However, Vincent et al. [20] and Grozdanovic et al. [11] reported a lack of NADPH-diaphorase staining in sympathetic ganglia, i.e. chain ganglia and celiac ganglion. Our data corroborate these studies in that numerous FG-labeled bladder perikarya were observed in the IMG, but NADPH-diaphorase reactivity was not present. Unlike sympathetic ganglia, numerous NADPH-diaphorase reactive perikarya are present in the M P G [6] and D R G [1]. In this study, numerous FG-labeled/ NADPH-diaphorase-positive bladder neurons were observed in both the M P G and D R G . These data suggest that if N O serves a physiological role in bladder function, it probably functions through the parasympathetic and/or sensory systems. Thornbury et al. [18] observed that nerve-evoked relaxation of the sheep bladder neck, i.e., internal smooth muscle sphincter, was mediated by NO, or a similar compound. Unfortunately the effects of N O on the detrusor muscle proper are unknown. However, the observation that a small number of intrinsic neurons and a sizeable population of postganglionic autonomic and sensory neurons are NADPH-diaphorase-positive suggest that NO may indeed serve a role in modulating bladder tone in the rat. The authors would like to thank Mr. Ben Han for his assistance with the photography. This study was supported by grants from the Paralyzed Veterans of America Spinal Cord Research Foundation and the Oklahoma Center for the Advancement of Science and Technology. 1 Aimi, Y., Fujimura, M., Vincent, S.R. and Kimura, H., Localization of NADPH-diaphorase-containingneurons in sensory ganglia of the rat, J. Comp. Neurol., 306 (1991)382-392. 2 Allescher,H.-D., Tougas, G., Vergara, P., Lu, S. and Daniel, E.E., Nitric oxide as a putative nonadrenergicnoncholinergicinhibitory transmitter in the caninepylorus in vivo,Am. J. Physiol.,262 (1992) G695-G702. 3 Buga, G.M. and Ignarro, L.J., Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery, Am. J. Physiol.,262 (1992)H973-H979. 4 Busch, P.A., Aronson, W.J., Buga, G.M., Rajfer, J. and Ignarro, L.J., Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum, J. Urol., 147 (1992) 1650-1655. 5 Crowe,R., Haven, A.J. and Burnstock, G., Intramural neurones of the guinea-pig urinary bladder: histochemicallocalizationof putative neurotransmittersin cultures and newborn animals, J. Auton. Nerv. Syst., 15 (1986) 31%339. 6 Dail, W.G., Galloway, B., Boubegaray, J. and Walton, G., Functional and histochemical evidence for the involvement of nitric oxidein regulationof penileerectiletissue, Soc.Neurosci. Abstr., 18 (1992) 128.

36 7 De Groat, W.C. and Booth, A.M., Physiology of the urinary bladder and urethra, Ann. Int. Med., 92 (1980) 312-315. 8 De Groat, W.C. and Saum, W.R., Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat, J. Physiol., 220 (1972) 297-314. 9 De Groat, W.C. and Steers, W.D., Neural control of the urinary bladder and sexual organs: experimental studies in animals. In R. Bannister (Ed.), Autonomic Failure, Oxford University Press, New York, 1988, pp. 196-222. 10 Gabella, G., Intramural neurons in the urinary bladder of guineapig, Cell Tissue Res., 261 (1990) 231-237. 11 Grozdanovic, Z., Baumgarten, H.G. and Bruning, G., Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225-235. 12 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. 13 Ignarro, L.J., Nitric oxide as the physiological mediator of penile erection, J. NIH Res., 4 (1992) 59~52. 14 Jancso, G. and Maggi, C.A., Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord, Brain Res., 418 (1987) 371 376.

15 Kannan, M.S. and Johnson, D.E., Nitric oxide mediates the neural nonadrenergic, noncholinergic relaxation of pig tracheal smooth muscle, Am. J. Physiol., 262 (1992) L511-L514. 16 Tabatabai, M.A., Booth, M. and de Groat, W.C., Morphological and electrophysiological properties of pelvic ganglion cells in the rat, Brain Res., 382 (1986) 61-70. 17 Tanaka, S. and Zukerman, C., A macroscopical study of the somatic visceral nerves innervating the male rat urogenital organs, Acta Anat., 56 (1981) 413414. 18 Thornbury, K.D., Hollywood, M.A. and McHale, N.G., Mediation by nitric oxide of neurogenic relaxation of the urinary bladder neck muscle in sheep, J. Physiol., 451 (1992) 133-144. 19 Sakuma, I., Togashi, H., Yoshioka, M., Saito, H., Yanagida, M., Tamura, M., Kobayashi, T., Yasuda, H., Gross, S.S. and Levi, R., N~-Methyl-L-arginine, an inhibitor of L-arginine-derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo, Circ. Res., 70 (1992) 607 611. 20 Vincent, S.R., Aimi, Y. and Kimura, H., The distribution of NADPH-diaphorase in the peripheral nervous system, Soc. Neurosci. Abstr., 15 (1989) 376.