Phillip Y.P. Jen

The distribution of vesicular acetylcholine transporter in the human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase.

Because doubt still remains concerning the distribution of nerves that are unequivocally cholinergic in the human genitourinary organs, we have used a specific marker, namely, an antibody to vesicular acetylcholine transporter (VAChT), to immunolabel cholinergic axons and cell bodies in specimens of urinary bladder, seminal vesicle, vas deferens, and prostate gland obtained from neonates and children post mortem. In addition some sections were double‐immunolabeled with VAChT and either neuropeptide Y (NPY) or nitric oxide synthase (NOS). The results demonstrated a rich cholinergic innervation to the muscle coat of the bladder body with a much less prominent, but nonetheless significant, cholinergic innervation to the smooth muscle components of the seminal vesicle, vas deferens, and prostate. Small ganglia were scattered throughout the detrusor muscle of the urinary bladder, approximately 75% of the intramural neurons being VAChT immunoreactive, whereas approximately 95% contained NPY and approximately 40% contained NOS. VAChT immunoreactivity was observed in 40% of neurons in ganglia scattered throughout the pelvic plexus. Almost all these cholinergic neurons contained NPY and approximately 65% contained NOS. Almost all the cholinergic nerve fibers throughout the genitourinary organs also contained NPY. Although NOS was sparse in the cholinergic nerves of the bladder body, it occurred in the majority of cholinergic nerves at the bladder neck and was also present in a proportion of the cholinergic nerves in the other organs examined. VAChT‐immunoreactive nerves were also observed in a sub‐epithelial location in all the organs examined, the majority containing NPY, whereas a small proportion contained NOS. Although doubt remains about the function of sub‐epithelial cholinergic nerves in the urinary bladder, the majority of similar nerves in the seminal vesicle, vas deferens, and prostate gland are considered to be secretomotor. Collectively these findings demonstrate that the cholinergic innervation of the male genitourinary system is well established in the neonate and child. Neurourol. Urodynam. 19:185–194, 2000.

NITRIC OXIDE SYNTHASE AND TYROSINE HYDROXYLASE ARE COLOCALIZED IN NERVES SUPPLYING THE POSTNATAL HUMAN MALE GENITOURINARY ORGANS

PURPOSE The objective of this study was to examine the distribution of nitric oxide synthase (NOS) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in nerve fibers supplying the human neonatal male genitourinary organs. MATERIALS AND METHODS An indirect double label immunofluorescence technique was employed on specimens obtained from infants and children at postmortem examination. RESULTS Many nerve fibers immunoreactive for both NOS and TH were observed in the muscle coat of the vas deferens and the seminal vesicle, within the fibromuscular stroma of the prostate gland and at the bladder neck, and also formed perivascular plexuses in each of these organs. Double-labeled nerves occurred less frequently in the intramural ureters and superficial trigone while similar nerves in the bladder body were relatively sparse. Numerous nerves immunoreactive for NOS but not TH were observed at the base of the epithelium of each organ examined. Four types of autonomic ganglion cell were observed in nearby pelvic ganglia: those which contained NOS and TH, those which contained NOS alone, those which contained TH alone and those which contained neither NOS nor TH. CONCLUSION The results indicate that many of the noradrenergic nerves as well as non-noradrenergic nerves supplying the male genitourinary organs have the capacity to synthesize nitric oxide (NO) and that NO may play a significant role in the autonomic control of both the urinary and genital organs in the postnatal human male.

The Distribution of Noradrenergic Nerves in the Human Lower Urinary Tract

The purpose of this presentation is to describe the distribution of noradrenergic nerves in the human genitourinary system. The techniques which have been employed include formaldehyde-induced fluorescence and immunocytochemical methods to demonstrate dopamine β-hydroxylase and tyrosine hydroxylase. These methods have been applied to human fetal, neonatal, infant, child and adult tissues removed either at post mortem examination or by surgical excision. The innervation of the fetal urinary bladder is well established by 13 weeks and, as in older specimens, the detrusor receives a sparse noradrenergic nerve supply. In contrast the smooth muscle of the terminal ureter is well supplied by this type of autonomic nerve. An additional incomplete muscle layer has been identified as a nomal component of the terminal ureter which is richly innervated by noradrenergic nerves. In some cases this muscle forms a complete collar which may be responsible for ureteric obstruction. By comparison with the detrusor, bladder neck smooth muscle receives a dense noradrenergic nerve supply particularly in the male. Unlike the detrusor, the structure and innervation of the vas deferens, seminal vesicle and prostate are poorly differentiated in the fetus. In the infant and child, the structure of the intramural smooth muscle of these organs remains immature although a rich noradrenergic nerve supply resembing the adult has been established in the fetus by 30 weeks. In the fetus, autonomic ganglia occur in association with noradrenaline rich paraganglia and surprisingly, with sensory nerve endings resembling pacinian corpuscles. Shortly after birth paraganglia are no longer associated with the autonomic ganglia of the genitourinary system. On the basis of size at least two types of autonomic neuron populate these autonomic ganglia. One type is relatively large and devoid of catecholamines but is closely associated with pericellular noradrenergic nerve fibres. The second type of neuron is small, contains noradrenaline and is arranged in clusters closely related to the capsule of the prostate gland. The significance of these observations will be considered with respect to the neurological control of the genitourinary system.

A double-label immunohistochemical study of intramural ganglia from the human male urinary bladder neck

Double‐label immunocytochemistry was used to investigate the colocalisation of various neuropeptides and the enzymes nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) in intramural ganglia of the human male urinary bladder neck and trigone. Postmortem specimens were obtained from 7 male infants and children ranging in age from 2 mo to 3 y who had died as a result of cot death or accidental trauma. On average 60% of the intramural neurons were non‐TH‐immunoreactive (‐IR) (i.e. presumptive cholinergic) and 40% were TH‐ and DbβH‐IR (i.e. noradrenergic). Within the non‐TH‐IR population, calcitonin gene‐related peptide (CGRP) was found in 65% of cells, neuropeptide Y (NPY) in 90%, nitric oxide synthase (NOS) in 45%, somatostatin (SOM) in 90%, and vasoactive intestinal polypeptide (VIP) in 40%. The corresponding values for the TH‐IR neurons were CGRP (54%), NPY (70%), NOS (58%), SOM (73%) and VIP (40%). All the observed bombesin (BOM)‐immunoreactivity was colocalised with TH while 90% of VIP and almost all the CGRP was colocalised with NPY. Less than 5% of neurons were immunoreactive for substance P (SP) or met‐enkephalin (m‐ENK) and some of these also contained TH. Varicose nerve fibres were seen in close proximity to some of the intramural neurons, the majority of such varicosities showing immunoreactivity to CGRP, VIP or TH. Less common were pericellular varicosities immunoreactive to NPY, SOM or SP. These results demonstrate the neurochemical heterogeneity of intramural neurons in the human bladder neck and provide indirect evidence for the complexity of the peripheral innervation of the human urinary bladder.

Co-localisation of tyrosine hydroxylase, nitric oxide synthase and neuropeptides in neurons of the human postnatal male pelvic ganglia

Double-label immunocytochemistry was used to investigate the co-localisation of neuropeptides and the enzyme nitric oxide synthase (NOS) with tyrosine hydroxylase (TH) in autonomic ganglia of the human postnatal male pelvic plexus. Postmortem specimens were obtained from six male infants and children ranging in age from 2 to 12 months who had died as a result of cot death or accidental trauma. On average, ganglia lying adjacent to the neck of the urinary bladder contained 45% of neurons which were TH-immunoreactive (-IR) while ganglia situated adjacent to the posterior and lateral aspects of the prostate gland contained 67% of neurons which were TH-IR. All the TH-IR neurons also contained dopamine beta-hydroxylase and were considered to be noradrenergic in type. On average, 61% of TH-IR neurons in bladder ganglia contained NOS, compared with 77% of non-TH-IR neurons (based on counts of over 1,000 cells in each case), while the percentages of TH- and non-TH-IR neurons containing neuropeptides were: calcitonin gene-related peptide (CGRP) (30%; 11%), neuropeptide Y (NPY) (66%; 92%), somatostatin (SOM) (70%; 29%), substance P (SP) (64%; 46%), vasoactive intestinal polypeptide (VIP) (64%; 83%). The equivalent values for TH- and non-TH-IR neurons in prostatic ganglia were NOS (38%; 59%), CGRP (55%; 18%), NPY (62%, 65%), SOM (14%, 20%), SP (13%, 8%) and VIP (42%; 82%). Varicose nerve fibers within the ganglia were seen forming pericellular arborizations around many of the ganglion cells, the most numerous containing TH-, CGRP-, NPY-, SOM- or VIP-immunoreactivity. Less common were pericellular varicosities containing SP-immunoreactivity while terminals containing NOS were not observed. No correlation could be detected between the peptide contents of the ganglion cells and of the associated pericellular terminals. However, the peptide content of the ganglion cells found in association with the urinary bladder and prostate gland correlates well with the previously documented coexistence of enzymes and neuropeptides in the intrinsic nerve fibers supplying these two regions of the human postnatal male genitourinary system.

Immunohistochemical localization of neuropeptide y and somatostatin in human fetal retina

The localization of neuropeptide Y and somatostatin in the retina of six prenatal human specimens was determined by the utilization of light microscopic immunoperoxidase and immunofluorescence methods. The age of the prenatal fetuses ranged from 15 to 40 weeks of gestation. At 15 weeks, round or pear-shaped neuropeptide Y-immunopositive cells were observed in the inner nuclear layer and somatostatin immunoreactivity was only detected in the ganglion cell layer. Positive neuropeptide Y ganglion cells were observed by 17 weeks of gestation and by 28 weeks neuropeptide Y-immunopositive cells were demonstrated in the ganglion cell layer and inner nuclear layer. At 26-28 weeks, neuropeptide Y-immunopositive cells exhibited approximately the same shape as at 15 weeks of gestation, but the appearance of one or two processes was detected extending from the somata. By 38-40 weeks of gestation, neuropeptide Y-immunopositive cells were detected in the ganglion cell layer, inner nuclear layer and an immunopositive fibrous configuration in the inner plexiform layer. During this same period (38-40 weeks), somatostatin-positive cells were located in the ganglion cell layer, inner nuclear layer and the inner segments layers. Due to the difficulty of obtaining fetal materials, the exact time of initiation in the expression of neuropeptide Y and somatostatin is presently hard to delineate; however, it is safe to state that the peptides appear early in development.

Structure and autonomic innervation of the human vas deferens: A review

The motor innervation of the smooth muscle coat of the human vas deferens is predominantly noradrenergic in type while a less dense and differently distributed presumptive cholinergic innervation is also in evidence, although the precise role of the latter is undetermined. Immunohistochemical studies have confirmed the presence of catecholamine‐synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DβH) in the majority of fine, varicose intramuscular nerves, about two‐thirds of which also contain neuropeptide Y (NPY). Minor populations of noradrenergic nerves contain enkephalin (ENK), galanin (GAL), somatostatin (SOM), or nitric oxide synthase (NOS). The presumptive cholinergic intramuscular nerves contain vasoactive intestinal polypeptide (VIP) and NPY. The subepithelial nerves of the vas deferens are assumed to have a secretomotor function and are rich in acetylcholinesterase and NPY, many also containing either VIP or NOS. The muscle coat of the human vas deferens is poorly differentiated until after birth, the intramuscular nerves in the fetus being relatively thick and non‐varicose. Development of a subepithelial nerve plexus lags behind that in the muscle coat but its density in the neonatal vas deferens resembles that seen in the adult. Observations on specimens of human vas deferens obtained at vasovasostomy carried out 1 to 15 years after vasectomy have shown a marked reduction in the density of noradrenergic nerves in the muscle coat of the testicular portion while that in the urethral portion remains unaltered. Furthermore, the subepithelial secretomotor nerves degenerate in the testicular portion. These long‐term changes in the pattern of innervation of the vas deferens consequent upon vasectomy may have profound effects upon the outcome of vasovasostomy with respect to subsequent sperm maturation, transport, and viability. Microsc. Res. Tech. 42:423–432, 1998.

Immunohistochemical characteristics of human paraganglion cells and sensory corpuscles associated with the urinary bladder. A developmental study in the male fetus, neonate and infant

Triple label immunohistochemistry was used to study the coexistence of the catecholamine‐synthesising enzymes dopamine beta‐hydroxylase (DBH) and tyrosine hydroxylase (TH) and several neuropeptides including neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene‐related peptide (CGRP), somatostatin (SOM) and galanin (GAL) as well as nitric oxide synthase (NOS) in developing pelvic paraganglion cells in a series of human male fetal, neonatal and infant specimens ranging in age from 13 wk of gestation to 3 y postnatal. 13–20 wk old fetal specimens possessed large clusters of paraganglion cells lying lateral to the urinary bladder and prostate gland which were intensely DBH‐immunoreactive (‐IR) but lacked TH, NOS and the neuropeptides investigated. With increasing fetal age small clusters of paraganglion cells were observed in the muscle coat of the urinary bladder. At 23 wk of gestation occasional paraganglion cells were NOS or NPY‐IR while at 26 wk of gestation the majority of paraganglion cells were TH‐IR and a few were SOM or GAL‐IR. Some postnatal paraganglia within the bladder musculature contained cells which were all VIP, SP or CGRP‐IR while others displayed coexistence of NOS and NPY, SP and CGRP, or NPY and VIP. The presence of NOS in certain paraganglion cells indicates their capacity to generate nitric oxide (NO). These results show that human paraganglion cells develop different phenotypes possibly dependent upon their location within the bladder wall. A delicate plexus of branching varicose nerves was observed in the fetal paraganglia which increased in density with increasing gestational age. The majority of these nerves were VIP‐IR while others were CGRP, SP, NPY, NOS or GAL‐IR. The presence of nerve terminals adjacent to the paraganglion cells implies a neural influence on the functional activity of the paraganglia. Some paraganglia in the late fetal and early postnatal specimens contained Timofeews sensory corpuscles, resembling pacinian corpuscles in their morphology. The central nerve fibre of these corpuscles displayed immunoreactivity for SP, CGRP and NOS, the latter indicating a possible role for NO in afferent transmission from the urinary bladder. In addition, a few corpuscles were penetrated by a noradrenergic nerve fibre immunoreactive for NPY and TH, which may have a modulatory role on the sensory receptor.

Tyrosine hydroxylase and vesicular acetylcholine transporter are coexpressed in a high proportion of intramural neurons of the human neonatal and child urinary bladder.

Autonomic ganglia of the human pelvic plexus contain sympathetic and parasympathetic neurons which innervate the internal reproductive organs and the lower urinary tract while the urinary bladder also receives innervation from small intramural ganglia embedded in the detrusor muscle. Previous studies have used the immunocytochemical demonstration of tyrosine hydroxylase (TH), either alone or in combination with dopamine beta-hydroxylase, to identify noradrenergic neurons in these ganglia. However until recently a reliable marker for cholinergic neurons in the human autonomic nervous system was not available since antibodies to choline acetyltransferase do not react in this tissue. The present immunohistochemical study has used an antibody to human vesicular acetylcholine transporter (VAChT) to identify cholinergic neurons in the pelvic plexus and intramural bladder ganglia in a series of specimens from human male neonates and children. Immunostaining for TH was also carried out on the same sections and the results showed that while the vast majority of pelvic ganglion neurons were either cholinergic or noradrenergic (as seen by the presence of VAChT or TH respectively), approximately 50% of the neurons in the intramural ganglia were labeled with both immunomarkers. The presence of TH in cholinergic neurons may be due to the immaturity of the tissues examined since previous data on intramural bladder ganglia in the adult have shown that a much smaller proportion of the neurons contain TH than was observed in the present study. It is concluded that the presence of TH alone cannot be regarded as a specific marker for noradrenergic neurons in the genitourinary system of the human neonate and child.

Colocalisation of neuropeptides, nitric oxide synthase and immunomarkers for catecholamines in nerve fibres of the adult human vas deferens

Single and double‐label immunofluorescence methods were used to determine the distribution and patterns of colocalisation of various neuropeptides and nitric oxide synthase (NOS) with the catecholamine synthesising enzymes tyrosine hydroxylase (TH) and dopamine‐beta‐hydroxylase (DβH) in nerve fibres within specimens of adult human vas deferens obtained at vasectomy (age range 28 to 83 y). Cholinergic nerve fibres were immunolabelled with an antiserum to vesicular acetylcholine transporter (VAChT). Using the general nerve marker protein gene product 9.5 (PGP) the density of intramural nerve fibres was found to be similar irrespective of age. Many of these axons, especially in the outer 2 muscle layers were TH and DbH‐immunoreactive (IR) and were thus confirmed as noradrenergic. Fewer such axons were seen in the inner longitudinal muscle layer. All the noradrenergic nerve fibres also displayed NPY‐immunoreactivity with minor populations containing galanin (GAL) or somatostatin (SOM). Nerve fibres lacking TH and DbH‐IR were immunoreactive for VAChT and were sparsely distributed throughout the 2 outer muscle layers but more numerous in the inner muscle layer. Nerves lacking TH and DbH were immunoreactive for NPY and some also contained NOS, VIP or CGRP. These results have been compared with those obtained previously from specimens of human neonatal and infant vas deferens where, in contrast to the present results, NOS and VIP were shown to be colocalised with TH in many of the intramuscular nerve fibres. It thus appears that NOS and VIP cease their coexistence with TH in intramuscular nerve fibres of the human vas deferens between the pre‐ and postpubertal states. In addition to the intramuscular nerve fibres a VAChT‐IR subepithelial nerve plexus occurs in the vas deferens and may control the secretory activity of the lining epithelium. Most of these subepithelial nerve fibres were immunoreactive for NPY and many also contained VIP while minor populations were immunoreactive for NOS, GAL, SOM or SP although fibres containing CGRP were not observed. The neuropeptide content of the subepithelial nerve plexus was similar to that observed in the infant, except for an increased density of VIP‐IR nerves, which may reflect greater activity of the lining epithelial cells in the adult vas deferens.