Preferential immunohistochemical localization of vasoactive intestinal polypeptide (VIP) in the sacral spinal cord of the cat: light and electron microscopic observations

In the present study we have employed immunoperoxidase techniques to investigate the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the spinal cord and sensory ganglia of the cat. The spinal distribution of VIP-containing neuronal processes was also compared with that of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) at lumbar, sacral, and coccygeal levels. At sacral levels, VIP was found to be contained in small and medium-sized primary sensory neurons and in dorsal rootlets. Deafferentation, by either ganglionectomy or dorsal rhizotomy, resulted in a nearly complete loss of VIP immunoreactivity in the spinal cord. The spinal distribution of VIP fibers and terminals was most dense and extensive in sacral segments. Forming a thin shell around the dorsal horn, collaterals, apparently originating from Lissauer's tract, projected either medially or laterally through lamina I. Laterally, many VIP axons terminated in lateral laminae V to VII. Others projected further through the neck of the dorsal horn to medial lamina V and the gray matter near the central canal. Medially, VIP axons descended through lamina I to expand into terminal fields in the posterior commissure and medial lamina V. At the ultrastructural level, VIP-like immunoreactivity was found in dense core vesicles within axonal enlargements containing both large dense core and smaller clear round vesicles. Synaptic connections were infrequently observed but, when encountered, were of the simple axodendritic type. The spinal distribution of VIP-containing fibers was remarkably similar to that reported for pelvic nerve visceral afferents, both in termination patterns within the spinal gray matter and in localization to the sacral cord. The density of SP-, SOM-, and CCK-containing fibers and terminals was constant at all levels examined (L4 to Co4). In marked contrast, the distribution of VIP fibers, much like that of pelvic nerve afferents, was mostly confined to sacral segments. Thus, although SP, SOM, and CCK may be contained within a population of sacral visceral afferents, they must be common to afferent systems in other segments as well. VIP, however, appears to be preferentially contained within pelvic visceral afferent fibers confined mostly to sacral segments.     

Immunohistochemical Characteristics and Distribution of Sensory Dorsal Root Ganglia Neurons Supplying the Urinary Bladder in the Male Pig

The study determined the distribution and immunohistochemical coding of the sensory neurons innervating the male pig urinary bladder. Retrograde tracer Fast Blue was injected bilaterally into the bladder trigone, base or dome. The presence of neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) and substance P (SP) were studied with immunofluorescence. Fast Blue-positive neurons were localized bilaterally in dorsal root ganglia from L1 to L6 and from S3 to S4 with specific differences regarding the injection site. The number of Fast Blue-positive neurons was higher in the right ganglia. Immunohistochemistry revealed that sensory neurons innervating the urinary bladder trigone, base and dome displayed immunoreactivities to CGRP, SP, NOS, GAL and SOM. Differences in the neuropeptide content were observed between the Fast Blue-positive neurons in lumbar and sacral ganglia. Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the urinary bladder trigone, base and dome.     

Expression of receptors for glial cell line-derived neurotrophic factor family ligands in sacral spinal cord reveals separate targets of pelvic afferent fibers

Nerve growth factor has been proposed to mediate many structural and chemical changes in bladder sensory neurons after injury or inflammation. We have examined the expression of receptors for the glial cell line-derived neurotrophic factor (GDNF) family within sensory terminals located in the sacral spinal cord and in bladder-projecting sacral dorsal root ganglion neurons of adult female Sprague-Dawley rats. Nerve fibers immunolabelled for GFRalpha1 (GDNF receptor), GFRalpha2 (neurturin receptor), or GFRalpha3 (artemin receptor) showed distinct distribution patterns in the spinal cord, suggesting separate populations of sensory fibers with different functions: GFRalpha1-labeled fibers were in outer lamina II and the lateral-collateral pathway and associated with autonomic interneurons and preganglionic neurons; GFRalpha2-labeled fibers were only in inner lamina II; GFRalpha3-labeled fibers were in lamina I, the lateral-collateral pathway, and areas surrounding dorsal groups of preganglionic neurons and associated interneurons. Immunofluorescence studies of retrogradely labelled bladder-projecting neurons in sacral dorsal root ganglia showed that approximately 25% expressed GFRalpha1 or GFRalpha3 immunoreactivity, the preferred receptors for GDNF and artemin, respectively. After cyclophosphamide-induced bladder inflammation, fluorescence intensity of GFRalpha1-positive fibers increased within the dorsal horn, but there was no change in the GFRalpha2- or GFRalpha3-positive fibers. These studies have shown that GDNF and artemin may target bladder sensory neurons and potentially mediate plasticity of sacral visceral afferent neurons following inflammation. Our results have also revealed three distinct subpopulations of sensory fibers within the sacral spinal cord, which have not been identified previously using other markers.     

Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat.

Unmyelinated sensory axons in the sacral spinal cord may play a role in bladder reflexes under certain pathological conditions. Previous data suggested vasoactive intestinal polypeptide (VIP) might be contained exclusively in sensory C-fibers, some of which innervate the bladder. This study was undertaken to describe the morphology of these VIP fibers in the sacral cord of the cat. VIP immunoreactivity was confined to unmyelinated axons observed at several levels of the sensory pathway including the dorsal root ganglia, dorsal roots, Lissauer's tract, and the lateral collateral pathway. A combination of light and electron microscopic observations showed VIP-immunoreactive fibers with labeled varicosities and synaptic terminals in laminae I, IIo, V, VII, and X. VIP-immunolabeled varicosities had a mean diameter of 1.6 microm (range = 0.11-7.4 microm, S.D. = 1.01, n = 311) with a small percentage (8%) being relatively large (3-7.4 microm). VIP varicosities contained a mixture of small clear vesicles (CLV) and large dense core vesicles (LDV). Although most varicosities contained a moderate number of LDVs (14.86 LDVs/microm2), some varicosities contained a large number of LDVs, whereas others contained very few. Varicosities that possessed synaptic specializations were classed as terminals and were divided into three morphological classes. Two of these resembled Gray's Type I terminal, whereas a third was similar to the Gray's Type II terminal. There was no consistent relationship between vesicle content of the terminal and the type of synaptic contact it possessed. This study shows that in the sacral spinal cord of the cat, VIP terminals originate only from C-fibers, terminate primarily in laminae I and V, and exhibit a variety of morphologies consistent with heterogeneous origins and functions of the lower urinary tract.     

The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive

The rat urinary bladder is innervated by neurons in dorsal root ganglia (DRG) that express the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), and a fraction of bladder afferents can bind the non-peptidergic marker isolectin B4 (IB4). We used histochemical binding and axonal tracing to identify the bladder afferents, and immunocytochemistry to determine the degree of colocalization of CGRP with IB4 in their cell bodies in DRG and in their central axons in the spinal cord. In the L6 DRG, about 60% of CGRP-positive neurons were also positive for IB4. In the spinal cord, IB4 and CGRP colocalized in fibers and terminals in the inner part of lamina II, the lateral collateral path, and the sacral parasympathetic nucleus (SPN). In SPN, the majority of IB4-positive fibers and terminals were also CGRP-positive. After injection of IB4 into the bladder wall, immunoreaction for IB4 was detected in SPN, but not in lamina II. These results suggest that most IB4-positive afferents from the bladder are also CGRP-positive, and that the distinction between peptidergic and non-peptidergic bladder afferents based on IB4 binding is of limited validity.     

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoreactive dynorphin B in sacral primary afferent fibers of the cat

Immunocytochemical analysis of the distribution of dynorphin B terminals in the sacral spinal cord of the cat revealed a pattern of staining very similar to that produced with antisera directed against the primary afferent derived, putative neurotransmitter, vasoactive intestinal polypeptide. Labeled axons and terminals were concentrated in lamina I and V and there was dense fiber staining in the tract of Lissauer. Of particular interest was the presence of immunoreactive axons in attached dorsal rootlets. To specifically focus on the possibility that some of the sacral primary afferent fibers are dynorphin-immunoreactive, we first tried to increase perikaryal labeling in the sacral dorsal root ganglia by topical treatment with colchicine. This did not produce immunoreactive labeling of cell bodies in the ganglia. Unilateral multiple dorsal rhizotomy (L5 to coccygeal 1), however, significantly decreased the staining of dynorphin-immunoreactive axons and terminals in the tract of Lissauer and in the dorsal horn of sacral segments ipsilateral to the deafferentation. No changes were detected in the lumbar cord. Finally, radioimmunoassay of caudal lumbar and sacral dorsal root ganglia was performed. Measurable immunoreactivity was found in all ganglia assayed, but, consistent with the histochemical analysis, sacral ganglia contained the highest concentration of immunoreactive dynorphin B. These data indicate that a significant component of the sacral spinal cord dynorphin terminal immunoreactivity derives from primary afferent fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons

Following sciatic nerve axotomy, vasoactive intestinal polypeptide (VIP) immunoreactivity increases dramatically in the central terminal areas of the nerve whereas other primary afferent neuropeptides are depleted. The contribution of the peripheral nerve to VIP increases in the spinal cord was investigated by performing sciatic nerve section alone, dorsal rhizotomy of the lumbar roots, axotomy and rhizotomy in combination or section of other peripheral nerves terminating in the same segments as the sciatic nerve. VIP, and for comparison, substance P (SP), cholecystokinin (CCK), somatostatin (SOM), were localized in the lumbar spinal cord and corresponding sensory ganglia using unlabeled antibody immunohistochemistry. After sciatic nerve section, SP, CCK and SOM were depleted in the lumbar dorsal horn whereas VIP increased. After rhizotomy alone all neuropeptide staining including VIP was depleted; axotomy followed by rhizotomy prodiced the same result. Axotomy of other peripheral nerves terminating in the lumbar cord increased the area of neuropeptide depletion but correspondingly increased the area of VIP staining. A large proportion of small and medium diameter dorsal root ganglion cells were stained for VIP after nerve section or axotomy but not after rhizotomy alone. A radical change in neuropeptide metabolism of dorsal root ganglion cells occurs after peripheral axotomy, in the form of a maked increase in VIP synthesis. An intact dorsal root is necessary for increases in VIP in the spinal cord indicating the primary afferent origin of the response.     

Immunohistochemical localization of seven different peptides in the human spinal cord

It is necessary to study the normal chemical contents in the human spinal cord in order to understand neurochemical changes that might occur under pathological conditions. In the present study, the comparative distribution of seven peptides was examined immunohistochemically in four levels (cervical, C; thoracic, T; lumbar, L; sacral, S) of the human spinal cord by means of the peroxidase-antiperoxidase technique. The peptides examined included bombesin (BOM), substance P (SP), cholecystokinin (CCK), somatostatin (SOM), methionine-enkephalin (M-ENK), vasoactive intestinal polypeptide (VIP), and thyrotropin releasing hormone (TRH). Among the seven peptides examined, four (BOM, CCK, SOM, and TRH) have never been described in the human spinal cord and the present work clearly demonstrates their existence in specific patterns. The terminals that were immunostained for BOM and CCK were localized in high concentration in the superficial dorsal horn (laminae I-II), in moderate amounts in the lateral part of laminae V and VII, and lesser amounts in the intermediate gray (lamina VII) and the dorsal part of the central gray (lamina X). Whereas BOM showed a similar distribution pattern at all spinal levels, CCK was mainly found in thoracic and lumbar levels. The SOM terminals were localized in the superficial dorsal horn (the highest density in lamina II but very few in lamina I), the intermediolateral cell column, intermediate gray, and central gray. This peptide was more widely distributed in the sacral cord with its terminal field extending into the ventral horn. The TRH terminals were mainly located in the ventral horn. Frequently, TRH terminals were seen adjacent to large ventral horn neurons. Furthermore, many neurons in the ventral and intermediate gray and Clarke's column demonstrated TRH immunoreactivity. The other three peptides (SP, M-ENK, and VIP) have been previously demonstrated in the human spinal cord and the present study confirmed their general spinal distribution with minor differences.     

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S₁–S₃ segments but not in adjacent segments (L₆–L₇ and Cx₁) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S₁–S₃), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L₇–S₂ dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat. © 1994 Wiley-Liss, Inc.     

Mechanisms underlying the pathogenesis of urinary bladder instability - new perspectives for the treatment of reflex incontinence

The urine storage ability of the urinary bladder is markedly impaired following inflammation of the urinary bladder and spinal cord injury because of a hyperexcitability of micturition reflexes. Using two rat models of inflammation-induced bladder overactivity and detrusor hyper-reflexia following spinal cord injury we investigated changes in the neuronal pathways to the urinary bladder which may underlie the development of this instability. Our results suggest that among the factors involved in inflammation-induced bladder instability are significant changes in the expression of the neuropeptides substance P, calcitonin gene-related peptide and galanin at the primary afferent level, as well as of the enzyme neuronal nitric oxide synthase (nNOS) at the afferent and postganglionic efferent level. In the lumbar and sacral spinal cord nNOS-immunoreactivity was depleted from dorsal horn neurones in both cystitis and spinal cord injured rats and from preganglionic parasympathetic neurones after spinal cord injury. Distension of the bladder in chronically spinalized rats elicited c-Fos expression in a significantly greater number of neurones throughout the lumbar and sacral segments than in rats with an intact neuraxis. Thus, under pathological conditions rather complicated changes in the synthesis of neuropeptides and nNOS occur at the primary afferent, spinal cord and postganglionic efferent level that together control the activity of the urinary bladder. Further mechanisms like unmasking of silent synapses and axonal sprouting in the spinal cord might further contribute to an increase in activity in micturition reflex pathways. Local cooling of the dorsal spinal cord at the level L6/S1 with temperatures between 14 and 20 °C proved a simple technique to control the unstable bladder and restore continence in both inflammation-induced detrusor overactivity and detrusor hyperreflexia following spinal cord injury. The effects of cooling are probably the result of a blockade of synaptic transmission within the dorsal cord which eliminates neuronal overactivity. Thus, local spinal cord cooling could offer a new method to treat bladder instability and reflex incontinence.     

Estrogen receptor-alpha and neural circuits to the spinal cord during pregnancy

Estrogen receptor immunoreactivity and mRNAs are present in spinal cord neurons in locations that are associated with sensory and autonomic innervation of female reproductive organs. The present study was undertaken to examine the expression of estrogen receptor-alpha in the spinal cord during different stages of pregnancy and to determine whether estrogen receptor-alpha-expressing neurons are related to uterine afferent nerves bringing information to the spinal cord at parturition. Immunohistochemistry showed estrogen receptor-alpha-immunoreactive neurons in the dorsal one-half of the spinal cord, i.e., dorsal horn, dorsal intermediate gray areas (dorsal commissural nucleus), and around the central canal and sacral parasympathetic autonomic nucleus of the lumbosacral spinal cord. Neurons in these areas corresponded topographically to the distribution of central processes of visceral primary afferent neurons (e.g., containing calcitonin gene-related peptide and substance P) that innervate and activate second-order spinal cord neurons (evidenced by their expression of Fos) at parturition. Western blots showed that estrogen receptor-alpha increases in the spinal cord, with a peak at day 20 of gestation, followed by a slight decrease by 2 days postpartum. These studies show that estrogen receptor-alpha is expressed by neurons in autonomic and sensory areas of the lumbosacral spinal cord that have connections with the female reproductive system and that the level of estrogen receptor-alpha changes over the course of pregnancy, which may follow profiles of steroid hormones. Many of these neurons may be involved in processing information related to reproductive organ function, changes during pregnancy, and relays to other CNS centers.     

Mechanisms underlying the pathogenesis of urinary bladder instability - new perspectives for the treatment of reflex incontinence

The urine storage ability of the urinary bladder is markedly impaired following inflammation of the urinary bladder and spinal cord injury because of a hyperexcitability of micturition reflexes. Using two rat models of inflammation-induced bladder overactivity and detrusor hyper-reflexia following spinal cord injury we investigated changes in the neuronal pathways to the urinary bladder which may underlie the development of this instability. Our results suggest that among the factors involved in inflammation-induced bladder instability are significant changes in the expression of the neuropeptides substance P, calcitonin gene-related peptide and galanin at the primary afferent level, as well as of the enzyme neuronal nitric oxide synthase (nNOS) at the afferent and postganglionic efferent level. In the lumbar and sacral spinal cord nNOS-immunoreactivity was depleted from dorsal horn neurones in both cystitis and spinal cord injured rats and from preganglionic parasympathetic neurones after spinal cord injury. Distension of the bladder in chronically spinalized rats elicited c-Fos expression in a significantly greater number of neurones throughout the lumbar and sacral segments than in rats with an intact neuraxis. Thus, under pathological conditions rather complicated changes in the synthesis of neuropeptides and nNOS occur at the primary afferent, spinal cord and postganglionic efferent level that together control the activity of the urinary bladder. Further mechanisms like unmasking of silent synapses and axonal sprouting in the spinal cord might further contribute to an increase in activity in micturition reflex pathways. Local cooling of the dorsal spinal cord at the level L6/S1 with temperatures between 14 and 20 degrees C proved a simple technique to control the unstable bladder and restore continence in both inflammation-induced detrusor overactivity and detrusor hyperreflexia following spinal cord injury. The effects of cooling are probably the result of a blockade of synaptic transmission within the dorsal cord which eliminates neuronal overactivity. Thus, local spinal cord cooling could offer a new method to treat bladder instability and reflex incontinence.     

Afferent fibers in the reproductive system and pelvic viscera of female rats: anterograde tracing and immunocytochemical studies

Innervation of the female reproductive system provides an important signal for a variety of neuroendocrine reflexes and behaviors in the female rat. Although some studies suggest that afferent feedback from the gonads is involved in the hypothalamic control of gonadal function and pituitary hormone release, the extent and function of afferent feedback from the gonads in these neuroendocrine reflexes has yet to be clarified. Deafferentation studies have provided only partial support for the afferent control of the gonads. Some studies even suggest functional asymmetries in the neural control of the gonads, but knowledge regarding the neuroanatomical substrate for these possible neurogonadal interactions remains incomplete. Studies with retrograde tract tracers indicate that the ovaries receive a substantial afferent supply from lower thoracic-upper lumbar dorsal root ganglia. Despite stringent precautions to prevent diffusion of tracers following injections into the ovary or related nerves, many of the retrogradely labeled cell bodies identified by these studies may represent an overestimation of the extent of afferent innervation. We have reexamined the afferent innervation of the female reproductive tract by means of the anterograde transport of horseradish peroxidase (HRP) from thoracic, lumbar and sacral dorsal root ganglion to pelvic visceral organs and have studied the effects of unilateral ganglionectomy on substance P containing fibers in the ovary, oviduct and uterus. The neuroanatomical results show that the T13 and L1 dorsal root ganglia provide major afferent innervation to the cranial portion of the reproductive tract and the L6 and S1 dorsal root ganglia provide primary afferent fibers to the caudal portion of the reproductive tract as well as the bladder, rectum and perineum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural analysis of enkephalinergic terminals in parasympathetic ganglia innervating the urinary bladder of the cat

Leucine enkephalin immunoreactivity was identified in axons and varicosities in parasympathetic ganglia located in the pelvic plexus and on the surface of the urinary bladder of the cat. Electron microscopic immunohistochemical studies revealed that varicosities containing leucine enkephalin exhibited large dense core vesicles and small, clear, spherical vesicles, which were similar to those found in cholinergic terminals. Leucine enkephalin immunoreactivity was primarily associated with large dense core vesicles. The varicosities formed axodendritic and axosomatic synapses with principal ganglion cells. Axoaxonic synapses were not detected. Some axosomatic enkephalinergic synapses were detected embedded within or invaginating the principal ganglion cells. Varicosities containing flattened and/or small dense core vesicles did not exhibit enkephalin immunoreactivity. Bladder ganglion cells identified by retrograde HRP tracing from the urinary bladder exhibited similar leucine enkephalinergic synapses. These observations, coupled with previous reports that leucine enkephalin is present in sacral preganglionic neurons and released by preganglionic nerve stimulation, suggest that leucine enkephalin and acetylcholine are cotransmitters stored and released from the same nerve terminals in bladder parasympathetic ganglia.     

Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder

We have investigated whether bladder afferent neurons are likely to be targets for circulating estrogens by mapping estrogen receptor (ER) distribution in lumbosacral dorsal root ganglia (DRG) of adult female rats. Sensory neurons innervating either the detrusor or trigone regions were identified by application of fluorescent retrograde tracer dyes to the bladder wall. Labelled neurons were classified by their immunoreactivity for either type of ER (ERalpha or ERbeta) and further compared with subpopulations of neurons containing substance P, calcitonin gene-related peptide and vanilloid receptor (a marker of polymodal nociceptors). Both ER types were expressed in numerous sensory neurons of either upper lumbar (L1/L2) or lower lumbar/sacral (L6/S1) ganglia and there was almost complete coexpression of ERalpha and ERbeta. ER-positive neurons were mainly small-medium size (18-25-microm diameter), indicating that they may be nociceptors and/or supply visceral targets. Most bladder-projecting neurons expressed ERs and the majority of these also expressed neuropeptides or vanilloid receptor. Afferent neurons supplying detrusor and trigone regions had similar immunohistochemical features. About a third of the bladder-projecting neurons expressed both ER and vanilloid receptor, suggesting a mechanism by which estrogens could influence bladder pain. The prevalence of different chemical classes of ER-positive bladder-projecting neurons was reflected throughout the entire population of neurons in dorsal root ganglia of these spinal levels, suggesting that neurons supplying other pelvic visceral targets may have similar chemical profiles. These results suggest that many functional classes of sensory neurons innervating the lower urinary tract are likely to be targets for circulating estrogens, including many nociceptor neurons. The coexistence of ERalpha and ERbeta suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system.     

Segmental analysis of neuropeptide concentrations in normal human spinal cord

We concurrently measured, by radioimmunoassay, levels of substance P (SP), somatostatin (SST), methionine-enkephalin (Met-Enk), cholecystokinin (CCK), peptide hystidyl-isoleucine (PHI), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the ventral and dorsal gray matter at each segment of the cervical, thoracic, lumbar, and sacral spinal cord, obtained within 6 hours of death from 4 subjects (ages 17 to 55) with no neurologic disease. Levels (pmol/g gray matter) of SP, SST, and Met-Enk throughout and PHI, VIP, and NPY in lumbar and sacral cord were significantly higher in dorsal than in ventral gray matter. PHI, VIP, and NPY were significantly higher in lumbar and especially sacral cord than in cervical and thoracic segments. In rats, a postmortem delay of up to 8 hours did not affect SP, Met-Enk, PHI, or NPY and decreased SST, CCK, and VIP levels. Thus, there is a characteristic profile of neuropeptide distribution in gray matter, which emphasizes the neurochemical heterogeneity along the rostrocaudal and dorsoventral extent of normal human spinal cord.     

Changes in the substance P-containing innervation of the lumbosacral spinal cord in male Wistar rats as a consequence of ageing

Quantitative image analysis was used to determine age-related changes in the substance P-containing innervation of autonomic and somatic nuclei in the lumbosacral spinal cord, which are associated with the control of micturition and sexual reflexes. In the upper lumbar segments (L1-L2), significant declines in the distribution density of substance P-containing processes were observed in the dorsal grey commissure, the intermediolateral cell column and the ventral horn. More caudally, at levels corresponding to L5 through S1, significant reductions were seen in the dorsal grey commissure and within the sacral parasympathetic nucleus. In contrast to these observations, the substance P-immunoreactive innervation of the dorsolateral nucleus remained robust in aged animals and was not significantly different from young adults. It is possible that these distinct age-related patterns of change in substance P-containing innervation, are reflected in the urinary/sexual dysfunction's in aged animals.     

The Role of Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptide in the Neural Pathways Controlling the Lower Urinary Tract

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in the neural pathways regulating the lower urinary tract. VIP-immunoreactivity (IR) is present in afferent and autonomic efferent neurons innervating the bladder and urethra, whereas PACAP-IR is present primarily in afferent neurons. Exogenously applied VIP relaxes bladder and urethral smooth muscle and excites parasympathetic neurons in bladder ganglia. PACAP relaxes bladder and urethral smooth muscle in some species (pig) but excites the smooth muscle in other species (mouse). Intrathecal administration of VIP in cats with an intact spinal cord suppresses reflex bladder activity, but intrathecal administration of VIP or PACAP in rats enhances bladder activity and suppresses urethral sphincter activity. PACAP has presynaptic facilitatory effects and direct excitatory effects on lumbosacral parasympathetic preganglionic neurons. Chronic spinal cord transection produces an expansion of VIP-IR (cats) and PACAP-IR (rats) in primary afferent axons in the lumbosacral spinal cord and unmasks spinal excitatory effects of VIP on bladder reflexes in cats. Intrathecal administration of PACAP6-38, a PAC1 receptor antagonist, reduces bladder hyperactivity in chronic spinal-cord-injured rats. These observations raise the possibility that VIP or PACAP have a role in the control of normal or abnormal voiding.     

Light and electron microscopic study of an avian pretectal nucleus, the lentiform nucleus of the mesencephalon, magnocellular division

The distribution of vasoactive intestinal polypeptide (VIP) was mapped by peroxidase immunocytochemistry in the spinal cords of seven Macaca fascicularis monkeys and two cats. The animals were perfusion fixed with different chemicals. Those that were perfused with either a Zamboni fixative or 5% acrolein had significantly greater immunoreactivity outside the sacral cords; those fixed with 4% paraformaldehyde had little in nonsacral regions. VIP-like immunoreactive (VIP) axons and terminals were found in the superficial dorsal horn, reticular nucleus of lamina V, intermediomedial nucleus, and lamina X at all levels from C2 to S4; a few axons and terminals were also seen in the ventral horn. Axons were found in Lissauer's tract at all levels, and axons appeared in the dorsolateral and ventrolateral white matter at midthoracic levels; in the lumbosacral cord the number and extent of axons in the lateral and ventral white matter increased progressively in a caudal direction. VIP neurons were identified in thoracic intermediate gray lateral to the central canal and in the intercalatus (IC) and intermediolateral (IML) nuclei. Electron microscopy of the VIP terminals in laminae I and II of the cervical cord revealed they contain small round vesicles and many large granular vesicles; some are glomerular terminals and most form asymmetrical synaptic contacts onto dendrites. These results indicate VIP is much more widely distributed in the spinal cord than previously thought; VIP may be associated with both visceral thoracic and lumbosacral afferents, and with other afferents in the cervical cord; VIP neurons are present in the thoracic intermediate gray; and VIP axons in the ventral and lateral white matter indicate that the spinal cord is supplied in part by VIP sources other than primary afferents.