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The presence and distribution of neuropeptide-containing nerves within bronchial surgical specimens has been investigated in bronchitic (n = 12) and in nonbronchitic subjects (n = 7). Lung tissue, obtained from patients undergoing thoracotomy for limited lung lesions, was processed immediately and analyzed for nerves using the streptavidin-biotin complex peroxidase method with antisera to the neural marker protein gene product 9.5 (PGP 9.5) and the neuropeptides vasoactive intestinal peptide (VIP), substance P (SP), calcitonin-gene related peptide (CGRP). There were no significant differences between the two groups with respect to the density of PGP 9.5-, SP-, or CGRP-positive nerves in both the locations assessed (smooth muscle layer and glands). The density of VIP-positive nerves was significantly higher in the glands of bronchitic than in nonbronchitic subjects. A negative relationship was found between the presence of airway inflammation, as indexed by mononuclear cell tissue infiltration, and the density of PGP 9.5-positive nerves in both smooth muscle and glands. Likewise, a relationship was found between the smoking history (packs/yr and age of onset of smoking) and the density of VIP-positive nerves in glands. These findings support a role for VIP in the hallmark of chronic bronchitis, i.e., sputum production.

Since pharmacological evidence indicates that nitric oxide (NO) operates in the control of uterine motility, we have studied the distribution of NADPH diaphorase and NO synthases in the rat uterus using histochemical and immunohistochemical methods. Numerous nerve fibers displayed NADPH diaphorase activity and immunoreactivity to antisera raised against neuronal NO synthase. Nerve fibers appeared in all stages of the estrous cycle and also after ovariectomy. NADPH diaphorase activity was also present in endothelia and cells dispersed in the different uterine layers. Most NADPH diaphorase-positive (ND) cells had eosinophilic granules with occasional cells expressing the ED1 macrophage-monocyte marker. Immunoreactivity for an inducible NO synthase was found in a small number of macrophage-like cells without NADPH diaphorase activity. Thus, ND cells may express another NO synthase isoform not detected by the available antisera. In normal cycling rats, ND cells were most abundant during proestrus, and their number further increased after estrogen treatment. ND cells were not observed after ovariectomy but were present after estrogen replacement therapy. ND cells could be involved in the estrogenic control of in vivo and in vitro uterine.

The POU family transcription factor Oct-2 was originally identified in B lymphocytes but has been shown to be expressed in neuronal cells, although it is absent in most other cell types. Cotransfection of Oct-2 expression vectors into nonneuronal cells with a tyrosine hydroxylase promoter/reporter plasmid suggests that Oct-2 can repress this promoter in this artificial situation. Here we report that reduction of endogenous Oct-2 levels in a neuronal cell line by an antisense approach results in an increase in endogenous tyrosine hydroxylase levels. In contrast, the level of the neuronal marker protein PGP9.5 remains unchanged in the antisense lines whereas that of the neuronal nitric oxide synthase decreases. Hence, the tyrosine hydroxylase gene is a natural target for repression by Oct-2 in neuronal cells. The significance of this effect is discussed in terms of the processes that regulate tyrosine hydroxylase gene expression and the role of Oct-2 in neuronal cells.

The present study provides light- and electron-microscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rough endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.

Nitric oxide is a free radical which is produced in the brain and is thought to be the first of a new class of neural messenger molecules. It is postulated to act by inducing an increase in cyclic guanosine monophosphate levels in target cells. The neuronal isoform of nitric oxide synthase, the enzyme responsible for the calcium-dependent synthesis of nitric oxide from L-arginine, has been purified from brain homogenate. Using a specific polyclonal antibody, we have localized brain nitric oxide synthase to the cytosol of discrete neuronal subpopulations and glial elements. These include non-pyramidal cells in the cerebral cortex, pyramidal and non-pyramidal cells of the hippocampus, aspiny neurons of the corpus striatum, basket, Purkinje and granule cells in the cerebellum and neurons of various brain stem nuclei. The localization of nitric oxide-producing neurons in morphologically different and neurochemically diverse cell types suggests a widespread neuromodulatory role for nitric oxide in the central nervous system of man.

The presence of nitric oxide synthase (NO-synthase), the enzyme responsible for the production of nitric oxide (NO) from L-arginine, is shown immunocytochemically in the intrinsic neurons of the human and porcine respiratory tract. NO-synthase immunoreactivity is demonstrated in a subpopulation of neurons of the microganglia present in the wall of the extra- and intrapulmonary bronchi as well as in the hilar region of the lung in relation to blood vessels. The immunostaining was also found in some nerve fibers of the respiratory nervous system. Human and porcine lung gave similar results. The possible involvement of NO in the nonadrenergic noncholinergic (NANC) nervous regulation of the lung is discussed.

A novel class of enteric neurons projecting directly from the rectal wall to the spinal cord, "rectospinal neurons", was investigated in rats by combined retrograde neuronal tracing, immunocytochemistry and electron microscopy. Rectospinal neurons were almost confined to myenteric ganglia of the distal rectum below the pelvic diaphragm and were labeled preferentially by injections into spinal cord segments L6/S1. Injections into more rostral spinal cord segments resulted in hardly any labeled enteric neurons. Dorsal and ventral rhizotomy experiments indicated an almost exclusive projection of rectospinal neurons through dorsal roots L6/S1 to the respective spinal cord segments. Among various peptides immunostained, vasoactive intestinal polypeptide and calcitonin gene-related peptide were selectively found in rectospinal neurons, which were also shown to contain calbindin, neurofilament protein- and peripherin-immunoreactivity. Vasoactive intestinal polypeptide- and calbindin-immunostaining were frequently co-localized in the same perikarya, while calcitonin gene-related peptide-immunoreactive rectospinal neurons probably represented a separate population. Neonatal capsaicin treatment did not significantly reduce the number of rectospinal neurons. Electron microscopy revealed synaptic contacts on the surface of rectospinal neurons. Taken together, these results establish rectospinal neurons as an anatomically and neurochemically distinct class of enteric neurons. Synaptic contacts on rectospinal neurons suggest that these neurons may function as a direct link from the enteric to the central nervous system, thus indicating that connections between these two networks are reciprocal.

The presence of nitric oxide synthase in spinal cord and dorsal root ganglia was investigated by immunohistochemistry using antibodies against the constitutive neuronal form of nitric oxide synthase (NOS). NOS immunoreactivity was present in both man and rat with similar distribution, being present in primary sensory neurons of dorsal root ganglia and their afferent terminals in dorsal horn of spinal cord. NOS immunoreactive interneurons were found in the superficial layer of the dorsal horn, around the central canal and in the intermediolateral cell column. NOS immunoreactivity was also present in numerous motoneurons in the ventral horn. The widespread distribution of NOS in both sensory and motor nervous system is indicative of the involvement of nitric oxide in different neural functions.

The nervous system may be actively involved in bone repair and in remodelling of callous tissue in bone fractures, as well as in the regulation of nociceptive impulses from the site of the trauma. The aim of this study was to assess the distribution and nature of the periosteal innervation of normal control bone and during bone healing subsequent to fracture of rat tibiae at seven, 14 and 21 days after experimental fracture using immunocytochemistry and image analysis quantification of the neuronal marker protein gene product 9.5 and sensory neuropeptide calcitonin gene-related peptide. At seven days, periosteal protein gene product 9.5- and calcitonin gene-related peptide-immunoreactive fibres showed dense ramifications and terminal sprouting. In addition to periosteum, the nerve fibres were found in the middle of the callus interspersed with inflammatory cells and penetrating into secondary minor fractures. At days 14 and 21 many tortuous nerves were found in the periosteum but not in mid callus. Image analysis quantification revealed a uniform increased proliferation of nerves after seven days. At 21 days, the intercept countings showed in excess of a three-fold increase of calcitonin gene-related peptide-immunoreactive nerve fibres compared with the normal control group (P > or = 0.0001) and were almost as numerous as protein gene product 9.5-immunoreactive fibres (P < 0.005). It is postulated that calcitonin gene-related peptide-containing sensory innervation may have a potential importance in the fracture vascular control, angiogenesis and osteogenesis in addition to a protective role against excessive fracture movement. The results are consistent with the neural involvement in bone growth and remodelling.

The effect of streptozotocin-induced diabetes on the production of vasoactive intestinal polypeptide messenger RNA in the myenteric neurones of 8 weeks diabetic rat intestine was examined using in situ hybridization. Total ganglion cell number per length (mm) of tissue section was measured using NADH diaphorase histochemistry. Although ganglion cells were more numerous in the control preparations compared with diabetic samples, a significantly greater number of vasoactive intestinal polypeptide messenger RNA-containing cells was detected in the diabetic tissues. These observations suggest that there is either a decrease in the breakdown of vasoactive intestinal polypeptide messenger RNA or an increase in its synthesis in myenteric neurones of diabetic rat intestine.

There is evidence that foetal body movements first occur at 6 weeks gestation, and that the reflex arc is functional at 8 weeks. This correlates with the detection of the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in spinal cord at 10 weeks gestation. However, the development of cutaneous neuropeptide-containing nerves is not well documented in humans. We have investigated the early appearance and distribution pattern of CGRP, SP, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), as well as those of the general neuronal marker protein gene product 9.5 (PGP) in various areas of foetal skin at different gestational ages. PGP-immunoreactive nerves were first seen in the subepidermal plexus at 6 weeks gestational age. Initially, the immunoreactive nerves are thick, club-shaped and distributed in the superficial dermis. Beaded adult-like fibres become more numerous only at later ages (10-12 weeks), and extend from this plexus to penetrate the epidermis. Histologically, the skin of the hand develops faster than that of other body areas and at 9 weeks, more PGP-immunoreactive nerves were seen in the palm than in the dorsum. Primitive sweat glands were first noted in axillary skin at 17 weeks, accompanied by a few PGP-immunoreactive nerves. Occasional, small CGRP-immunoreactive fibres were first noticed in the dermis at 7 weeks, but it was at 17 weeks that the presence of this neuropeptide was unequivocal in the subepidermal plexus. Sparse VIP-, SP- and NPY-immunoreactive fibres were not found until 16-17 weeks gestation, when they were seen in the dermis and around small blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

The SmN protein is closely related to the ubiquitously expressed SmB and B' RNA splicing proteins but is expressed in only a limited range of tissues and cell types. The expression of SmN in a range of neuronal and non-neuronal cell lines correlates with their ability to splice the calcitonin/CGRP transcript to produce the mRNA encoding CGRP rather than that encoding calcitonin. Moreover, the SmN mRNA shows a widespread distribution within the brain and spinal ganglia being present in neuronal cells in all regions which naturally produce CGRP as well as in those areas which do not naturally express the calcitonin/CGRP gene but which can correctly splice the CGRP mRNA in transgenic mice expressing the calcitonin/CGRP gene in all cell types. Interestingly however the mRNA encoding SmN is also found in a few areas of the brain which can only carry out calcitonin-specific splicing in transgenic mice, such as the Purkinje layer of the cerebellum and the inferior colliculus. The possible role of SmN in the regulation of splicing in neuronal cells is discussed in the light of these results.

The use of the polymerase chain reaction in conjunction with degenerate oligonucleotides has allowed the isolation of cDNA clones derived from each of the POU family transcription factors expressed in the proliferating ND7 neuronal cell line. In addition to the previously characterized Oct-1, Oct-2 and Brn-3 factors, ND7 cells have been shown by this means to express a novel POU factor. This factor is closely related to Brn-3 but differs at seven amino acids in the POU domain, one of which is located in a region which is critical for protein-protein interactions between different POU proteins. Like Brn-3, the novel factor is expressed at high levels in embryonic brain and declines in abundance during neuronal development. In contrast to Brn-3 however, it is absent in mature sensory neurons and its expression declines during the in vitro differentiation of ND7 cells to a non-dividing phenotype whilst Brn-3 expression increases. The significance of these distinct but overlapping expression patterns is discussed in terms of the possible role of these two factors in regulating neuronal gene expression.

Deer antler is a unique mineralized tissue which can produce very high growth rates of > 1 cm/day in large species. On completion of antler growth, the dermal tissues which cover the antler are shed and the underlying calcified tissue dies. After several months the old antler is discarded and growth of a new one begins. It is known that deer antlers are sensitive to touch and are innervated. The major aims of this study were to identify and localize by immunohistochemical techniques the type of innervation present, and to find out whether nerve fibres could exhibit growth rates comparable to those of antler. We have taken tissue sections from the tip and shaft of growing Red deer (Cervus elaphus) antlers at three stages of development; shortly after the initiation of regrowth, the rapid growth phase, and near the end of growth. Incubation of tissue sections with antisera to protein gene product 9.5 (a neural cytoplasmic protein), neurofilament triplet proteins (a neural cytoskeletal protein), substance P and calcitonin gene-related peptide (both of which are present in and synthesized by sensory neurons) showed the presence of immunoreactive nerve fibres in dermal, deep connective and perichondrial/periosteal tissues at all stages of antler growth. The sparse distribution of vasoactive intestinal polypeptide-like immunoreactivity was found in dermal tissue only at the earliest stage of antler development. Nerve fibres immunoreactive to neuropeptide Y, C-flanking peptide of neuropeptide Y and tyrosine hydroxylase, all present in postganglionic sympathetic nerves, were not observed at any stage of antler growth. Nerves expressing immunoreactivity for any of the neural markers or peptides employed could not be found in cartilage, osteoid or bone. These results show that antlers are innervated mainly by sensory nerves and that nerves can attain the exceptionally high growth rates found in regenerating antler.

By 8 wk gestation, the human fetal oesophagus is identifiable as a hollow epithelium-lined tube with primitive nerve and muscle precursors present. From 8-16 wk gestation, the muscle layers and innervation mature until fetal swallowing commences at 16 wk. This study examines quantitatively the development and maturation of nerve fibres and cell bodies within the oesophagus using histochemistry. Oesophageal samples (n = 35) from 8 wk gestation to 28 months of age and adults (n = 3) were immunostained using antisera for the general nerve marker, protein gene product (PGP 9.5), the glial tissue marker S100, and the synaptic vesicle protein synaptophysin (p38). Histochemical staining for NADH diaphorase enzyme activity was also used to identify neurons. Computer-assisted image analysis of the muscularis externa permitted detailed quantification of cell size, nerve density and myenteric (plexus) fraction. At 8 wk gestation, PGP and synaptophysin were present in immature neurons throughout the cytoplasm, but from 10 wk synaptophysin was localised solely at nerve synapses. S100 immunoreactivity was also detected from 8 wk gestation onwards and was confined to glial tissue. Nerve cell size increased with maturation from 6 microns at 8 wk gestation to 20 microns at term and 21 microns at 28 months. The numbers of cells, nerve density (% area occupied by nerves throughout section) and myenteric fraction (% area occupied by ganglion cells and nerve fibres within the myenteric plexus) all peaked at 16-20 wk gestation and, whereas the number and density then fell towards adult levels, the myenteric fraction fell during the late second trimester and became constant from 30 wk gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to assess the relationship of neuropeptide nerves and inflammatory leukocytes in PVG rats with adjuvant-induced arthritis. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and inflammatory leukocytes were studied, using peroxidase (ABC) and/or alkaline phosphatase (APAAP) staining. Inflamed synovial tissue proper was infiltrated with neutrophils, ED1 macrophages and focal accumulations of CD2 T lymphocytes. In such tissue, the relationship between peptide-immunoreactive nerves and inflammatory cells was such that substance P and CGRP nerves were absent in heavily infiltrated villous synovial tissue, whereas healthy synovial tissue and non-inflammatory areas in adjuvant arthritic rats were innervated by substance P and CGRP nerves close to normal synovial tissue resident cells. In order to elucidate an eventual mechanism for lost immunoreactivity, healthy synovial tissue was exposed to chymotrypsin or oxygen derived free radicals (ODFR) in vitro. The former treatment caused total loss of immunoreactivity. These findings suggest that neuropeptides and neuropeptide containing nerves may be destroyed by locally produced proteolytic enzymes and various reactive oxygen species in the vicinity of inflammatory cells.

The increased expression of vasoactive intestinal polypeptide (VIP) in injured peripheral neurons was studied. In contrast to substance P, there was a marked increase, and maintained fast axonal transport, of VIP in rat sciatic nerve after peripheral axotomy. Local capsaicin application to the nerve trunk failed to inhibit the injury-induced VIP increase, and capsaicin even increased VIP levels when applied locally to uninjured nerves. Pharmacological sympathectomy showed that some of the peripheral VIP increase may occur in post-ganglionic sympathetic fibres. The VIP increase after injury appeared unaffected in the mf mutant rat, in spite of its loss of lumbar dorsal root ganglion cells. VIP-staining fibres in the epi- and peri-neurium and perivascular plexuses of sciatic nerve showed an increase in number in parallel with the changes of the nerve VIP content. These findings suggest that sensory and sympathetic nerve fibres expressing VIP after injury play a role in the regulation of blood flow to nerves, and in the pathophysiological processes in nerve and dorsal spinal cord which follow peripheral nerve injury.

The anatomical basis of the peptidergic neural control of the human colon is largely unknown. In this study, in vitro retrograde tracing methods have been used on fresh human colon to determine the projection pathways of the enteric nerves and, in particular, those containing vasoactive intestinal polypeptide, one of the most abundant and potent of the gut neuropeptides. Two components of the submucous plexus were identified, the inner one projecting to the lamina propria, and the outer to the circular muscle. The lengths of projections within the submucous plexus were up to 5-14 mm in all directions. Myenteric ganglion cells projected to both longitudinal and circular muscles, for distances of up to only 5 mm. The subpopulation of nerves containing vasoactive intestinal polypeptide arose mainly from the submucous plexus and projected up to 6.5 mm anally, 5 mm orally, and 14 mm within the submucous layer to the mucosa or circular muscle. These findings provide entirely new data on the neuroanatomy of the human colon and may help in the understanding of the neural control of colonic secretion and motility.

By means of antisera to cytoplasmic components of nerve fibres and neuropeptides which are known to be present in sensory or sympathetic nerves we have examined the distribution of both total and different types of nerve fibres in normal and inflamed human synovial tissue. Samples of synovia were obtained at surgery from five normal and five rheumatoid patients (age range 10-77 years). In order to map the overall neural innervation of the synovium, antiserum to the general neuronal marker protein gene product 9.5 was employed. Substance P and calcitonin gene-related peptide antisera were employed to identify sensory fibres and antisera to the C-flanking peptide of neuropeptide Y to distinguish sympathetic nerves. In normal synovium protein gene product 9.5-immunoreactive fibres were numerous, in particular, the vasculature was densely innervated. Free protein gene product 9.5-immunoreactive fibres were less numerous but were present in all synovia examined, and in many cases these extended to the intimal layer. Neuropeptide immunostaining was predominantly found in perivascular networks. Fibres immunoreactive for the C-flanking peptide of neuropeptide Y were exclusively located around blood vessels whereas free fibres were immunoreactive for substance P or calcitonin gene-related peptide. As with free protein gene product 9.5-immunoreactive fibres, fibres expressing substance P or calcitonin gene-related peptide immunoreactivity were often seen in the intimal cell layer. In rheumatoid arthritis a similar innervation to that seen in normal synovium was apparent in the deep tissue but fibres immunoreactive for protein gene product 9.5, the C-flanking peptide of neuropeptide Y, substance P or calcitonin gene-related peptide were not visible in the more superficial tissues or the intimal cell layer. In addition, immunostaining of neuropeptides in the deep tissue was weaker in the diseased tissues than in normal controls. The data unequivocally demonstrate that synovial tissues are richly innervated and confirm the presence of both sensory and sympathetic nerves. The absence of nerves which innervate the superficial synovium in rheumatoid arthritis might suggest that there is increased release of substance P, calcitonin gene-related peptide and the C-flanking peptide of neuropeptide Y, reducing the stores in the nerves to levels below that detectable by immunocytochemistry. However, since protein gene product 9.5-immunoreactive nerves were not seen in the inflamed tissue it is probable that synovial growth outflanks neural growth and consequently as the disease progresses neural structures become restricted to deeper tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurons identified by their immunoreactivity with antisera against neuropeptide Y (NPY) were studied in three selected areas of the cerebral cortex in brains from controls and in senile dementia of the Alzheimer type (ATD). Changes were more profound in temporal cortex than in parietal cortex, and more severe in parietal cortex than in frontal cortex, paralleling the severity of neuritic plaque formation and incidence of neurofibrillary tangles in these regions. NPY-i neurons became distorted, with enlarged misshapen cell somata and reduced, thickened, and gnarled dendrites. There was a sharp reduction in the extensiveness and delicacy of the axonal plexus; the reorganized axons were haphazard compared to the normal symmetry of these fibers. Besides the alteration in form and sizes, there were also appreciably fewer cells. Nevertheless, the NPY population is not eliminated. Double-label studies of NPY-i and thioflavin indicate that NPY-i fibers can participate in neuritic plaque formation although not all neuritic plaques contained NPY-i axons and not all NPY-i axons were associated with plaques. The surviving NPY cells were evident in all cortices examined, thus giving rise to the speculation that these peptide neurons may have unusual survival and reorganization potential even in terminal neurological disease.

VIP is present in the genitourinary system of man and animals. In man the highest concentrations are found in the penis, the uterus and vagina and in the urinary bladder. VIP nerves heavily innervate the erectile tissue of the male external genitalia, the uterine smooth muscle and blood vessels, the seromucous glands of the cervix, and the lamina propria and vaginal epithelium. In the urinary bladder, VIP nerves are located beneath the transitional epithelium, in the lamina propria and in the smooth muscle. Other areas well innervated by VIP nerves include the prostate, seminal vesicles and vasa deferentia. Chemical (phenol- and 6-OHDA) or surgical (hypogastric or pelvic nerve section) extrinsic denervation fail to deplete the genitourinary system of its VIP content, supporting the view that VIP-containing nerves originate from local ganglion cells. Indeed, neuronal cell bodies containing VIP are seen in the paracervical ganglia of the female genitalia, the para- or intramural bladder ganglia and scattered through the base of the cavernosum body, the neck of the bladder and the prostate. The finding of elevated levels of VIP in the local circulation after induced penile erection in man and mammals and the ability of VIP to relax the detrusor muscle of the bladder suggests that the peptide may be involved in penile erection and bladder relaxation, as does the marked VIP depletion in the penis or bladder in patients suffering from diabetic impotence or bladder instability.

The innervation of the urinary bladder is known to include a considerable number of nerves containing vasoactive intestinal polypeptide (VIP). The origin of such nerves in the bladder of rat was investigated in this study using the methods of immunocytochemistry and radioimmunoassay combined with surgical sectioning of the hypogastric and/or pelvic nerves to the bladder. Eight days after pelvic nerve sectioning proximal to the main pelvic ganglion, VIP-immunoreactive nerves and VIP content were markedly increased from the level in the sham-operated rat bladder. Sectioning of hypogastric or both nerve pathways led to a less significant increase. It was therefore postulated that the majority of VIP-immunoreactive nerves originate from ganglia located either close to the bladder or within the bladder wall. It is interesting that in these experiments the VIP content of the bladder nerves is inversely related to the changes in motility that would be expected to result from the nerve sections.

Hyperplasia of endocrine cells in the lung of the adult rat exposed to asbestos has only been characterised so far by electron microscopy as there is a lack of reliable staining techniques for their demonstration at light microscopical level. Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase has recently been shown to be present in lung endocrine cells. In this study we reveal a marked endocrine cell hyperplasia at light microscopical level in the lungs of adult rats exposed to asbestos using antibodies to NSE. Very large groups of NSE-immunoreactive cells (20-80) were only observed in the lungs of rats exposed to asbestos for 12 months. In addition smaller groups of cells (2-10) known to be present normally and to decrease with age, were rarely noted in the controls but were frequently detected in the treated rats. Immunoreactive NSE is therefore a very good marker for endocrine cell hyperplasia and thus of early neoplastic changes.

The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.

Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A2-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60-100 nm diameter), electron-dense, membrane-bounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immuno-staining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.

We have demonstrated that calcitonin gene-related peptide (CGRP) immunoreactivity is widely distributed in cardiac and perivascular nerves of the guinea pig and rat. In the guinea pig the number and distribution of CGRP-immunoreactive nerve fibres closely paralleled that of fibres containing substance P, the two immunoreactivities being found invariably to coexist in the same perivascular networks and terminals. In the rat, CGRP-immunoreactive cardiovascular nerves had a similar distribution to those containing substance P, but in contrast to the guinea pig the former were far more numerous. Marked regional variations were observed in the density of the CGRP-immunoreactive innervation in both species. The CGRP-immunoreactive content of tissue extracts was in close agreement with the immunocytochemical findings, the highest levels of CGRP occurring in the mesenteric artery (guinea pig and rat) and inferior vena cava (guinea pig). Following capsaicin treatment of adult guinea pigs and neonatal rats, there was a significant loss of CGRP-immunoreactive nerves in the two species. In the guinea pig, substance P-and CGRP-immunostained fibres were depleted to a similar extent, throughout the cardiovascular system. However, the loss of rat CGRP-immunoreactive nerves was dose-dependent and displayed considerable variation, some perivascular nerve networks appearing less susceptible than others to the action of capsaicin. The results suggest that there may be species differences in the sensitivity of CGRP-containing nerves to capsaicin treatment, but at least the majority of CGRP-immunoreactive cardiovascular nerves may be presumed to be sensory in origin.

Neuron-specific enolase (NSE) is a neuronal form of the glycolytic enzyme enolase, which was first found in extracts of brain tissue, and was later shown to be present in APUD (amine precursor uptake and decarboxylation) cells and neurons of the diffuse neuroendocrine system but not in other peripheral cells. 90 neuroendocrine neoplasias (APUDomas) (including islet-cell tumours, phaeochromocytomas, medullary thyroid carcinomas, oat-cell tumours, and APUDomas of the gut, pancreas, and lung) reacted strongly with antisera to NSE. In addition, large amounts of the enzyme were found by radioimmunoassay in the tumours (mean 1626 +/- 479 SEM ng of NSE/mg protein), whereas control non-endocrine neoplasias contained less than 15 ng NSE/mg protein. Thus NSE, a specific enzyme produced in the neural and endocrine systems, was found to be produced in considerable quantities by all types of APUDomas but not in any non-endocrine tumours. NSE seems to be a useful and easily detected marker which may be used to distinguish endocrine from nonendocrine neoplasias. Clinical detection of endocrine tumours is difficult and such tumours are often missed. Use of NSE as a marker may avoid this.

The regional distributions of neuropeptide Y (NPY), peptide histidine-methionine (PHM), and vasoactive intestinal polypeptide (VIP) immunoreactivities in the human female genital tract have been estimated by specific radioimmunoassays, and their molecular forms determined by chromatography. The localisation and distribution of these three peptides was carried out by immunocytochemistry. The vagina and cervix contain high concentrations of NPY- and VIP-immunoreactive nerves, mainly localised around the vascular and nonvascular smooth muscle. VIP-containing nerves were, in addition, seen beneath the cervical and, in particular, the vaginal epithelium. A comparatively high level of immunoreactive NPY is found in the fallopian tube, mainly around the circular muscle coat. There is evidence that VIP is a neurotransmitter in the female genital tract, and these results suggest a similar role for NPY and PHM.

NSE concentrations were measured by radioimmunoassay in the main separated layers of the human gastrointestinal tract. At all levels, a similar pattern of distribution of this protein was found, primarily parallel to that of nerve elements. Lower amounts of NSE were detected in the separated mucosal epithelium, containing the endocrine cells.

The frontal, parietal, and temporal cortices in normal human brains (Brodmann areas 10, 7a, 7b, and 21) are well endowed with numerous neurons, identifiable by immunoreactivity with antisera against the 36-amino acid brain peptide neuropeptide Y (NPY). These neurons with rare exception are small, intracortical, nonspiny neurons, 12-20 microns in somatic size, with long slender dendrites and exuberant axon plexuses exhibiting finely beaded varicosities. The cells are rarest in layers I and II, are found with frequency in the lower cortical layers (IV-VI) and in significant numbers in the subcortical white matter. Within the cortex the axonal plexuses of these peptide neurons rise straight up into the upper cortical layers or descend deep into the white matter. In layers I and II, numerous other lengthy axons, some possibly of extracortical afferent origin, run along the pial surface at right angles to the axial ones running perpendicular to the cortex. This endowment of peptide neurons and their processes forms a rich network in the cerebral cortex, relating with one another in complex fashion within palisades of terminals as well as with the other cortical neurons not labeled by these methods. It remains to be shown what functions these NPY neurons have individually and in their remarkable networks, and how they are altered in neurological disease.

Lung innervation has been studied in the past by methylene blue staining and silver impregnation and more recently by histochemical methods. These techniques give only a partial picture of the total innervation. We have delineated the innervation of the lung in man and three other mammalian species by immunostaining with antibodies to two new markers of nervous tissue. These markers are neurone-specific enolase (NSE), an enzyme present in nerve cells in both the central and the peripheral nervous systems, and S-100, a protein found in glial cells. Throughout the respiratory tract NSE was localised in ganglion cells and nerve fibres in all species examined, while S-100 was found in the supporting glial cells of ganglia and in Schwann cells of peripheral nerves. The distribution of NSE immunoreactivity in serial sections was compared with that of acetylcholinesterase-containing, noradrenergic, and peptide-containing nerves. In all areas NSE was found to be a specific marker for all three types of nerves. Thus these two antibodies provide an effective histological means of examining both the neuronal and the non-neuronal components of the lung innervation and should be of value in investigating this system in lung disease.

The localization of endothelin 1 mRNA and endothelin-like immunoreactivity was investigated in samples of neurologically normal nervous system tissue from 10 adults by using in situ hybridization and immunocytochemistry. Tissue sections of spinal cord and dorsal root ganglia were hybridized with an 35S-radiolabeled endothelin 1 complementary RNA probe. Autoradiograms showed labeled neurons in the spinal cord (laminae IV-VI and many motoneurons) and numerous small and large neurons in the dorsal root ganglia. Endothelin 1 transcripts were also found in association with the endothelial layer of some blood vessels in the white matter of the spinal cord. A similar distribution of immunoreactivity was seen using three antisera to endothelin 1, but fewer cells were immunostained than were labeled after the hybridization. Two other endothelin 1 antisera immunostained the endothelial lining of blood vessels in the spinal cord white matter but not neurons. In the ganglia, endothelin 1 transcripts were localized to all cells expressing beta-preprotachykinin and most expressing calcitonin gene-related peptide mRNAs; in the majority of the motoneurons, coexistence of endothelin 1 and calcitonin gene-related peptide mRNAs was noted. There was a similar pattern of coexistence for respective peptide immunoreactivities in the ganglia and spinal cord. The expression of endothelin 1 mRNA in distinct neuronal cell types suggests that this peptide plays a part in neural transmission/modulation and adds a further dimension to the known vascular-related actions of endothelin 1.

Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase, has been established by immunocytochemical means as a marker of morphological and functional maturation in central neurons and appears late in development. However, little is known about the presence of NSE in developing peripheral neurons and endocrine cells and its relationship to the development of classical neurotransmitters and peptides. We therefore investigated the appearance of NSE immunoreactivity in nerves and mucosal endocrine cells of the human respiratory tract in foetal, neonatal and adult life. NSE was found to be present in neuroblasts, nerve fibres and endocrine cells from the earliest period of gestation examined (8 weeks), before the appearance of acetylcholinesterase activity (10-12 weeks), dopamine-beta-hydroxylase (20 weeks), vasoactive intestinal polypeptide (20 weeks) or calcitonin (20 weeks). Bombesin-like immunoreactivity was found in a small proportion of mucosal endocrine cells as early as eight weeks in the foetal respiratory tract. These findings indicate that unlike central neurons and their processes, peripheral neurons of the lung contain NSE immunoreactivity well before full maturation and establishment of synaptic contact with end organs.

A combination of in situ hybridization for neuropeptide Y mRNA that used a 32P-labeled complementary RNA probe and immunocytochemistry with polyclonal antibodies against neuropeptide Y were applied to human cortical brain samples to simultaneously localize neuropeptide Y and its mRNA. These two techniques allowed simultaneous identification of neuropeptide Y gene expression and peptide storage in single neurons of the human brain.

Merkel cells are specialized skin receptor cells, characterized by their particular location in the epidermis and close association with nerve terminals. Although they can be distinguished ultrastructurally by their small, electron-dense secretory granules, there is no specific and reliable method for identifying them by light microscopy. Using antibodies to neuron-specific enolase (NSE), the authors have shown sparsely distributed groups of specifically immunostained cells and associated nerve terminals in the nose skin of cats and rats. These cells were easily distinguished from other epithelial cell types, including melanocytes and Langerhans cells and had all the morphologic features of Merkel cells and their so-called neurite complexes, including the characteristic cytoplasmic secretory granules (60 nm in diameter). NSE immunostaining is a simple and reliable method for the specific light-microscopic staining of Merkel cells and provides further evidence for NSE as a marker for the diffuse neuroendocrine system.

Immunocytochemistry against neuropeptides contained within primary afferent neurons was used to study the morphology and distribution of intraepidermal free nerve endings in cat facial skin. Calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity was found in similar intraepidermal nerve endings of cat glabrous and hairy skin epithelia. The greatest density of immunoreactive intraepidermal nerve endings was located in the nose epidermis. Small limited areas with immunoreactive intraepidermal nerve endings were also found in facial hairy skin.

Using the methods of immunocytochemistry and radioimmunoassay, five peptides (vasoactive intestinal polypeptide (VIP), substance P, somatostatin, met-enkephalin, and bombesin) have been found in the gall bladder and the biliary tracts of guinea pig and each of them possesses a characteristic distribution pattern. Networks of nerves containing each peptide were found in the smooth muscle, around blood vessels and, occasionally, in the lamina propria. The distribution of the peptide immunoreactive nerves in the gall bladder and biliary tract is similar to those found in the gut. Vasoactive intestinal polypeptide (11 +/- 1.5 pmol/g in the sphincters, mean +/- SEM) and substance P (21.5 +/- 1.8 pmol/g in the common bile duct) were found to be the most abundant peptides and a few VIP and substance P immunoreactive neurones were localised in the ganglionated plexus. Bombesin immunoreactive nerves were mainly seen in the sphincter of Oddi, where the mean concentration of extractable bombesin was 14.6 +/- 2 pmol/g. Somatostatin immunoreactive mucosal endocrine cells were identified in the epithelium of the common bile duct and the sphincter. The extractable somatostatin in these regions were 76 +/- 19 pmol/g and 162 +/- 30 pmol/g respectively.

The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptide-containing nerves and myocardial cells producing ANP.

By using a specific antibody against the 29 amino-acid peptide galanin (Gal) with light and electron microscopic immunocytochemistry, we have studied the distribution of Gal immunoreactivity in the posterior hypothalamic magnocellular neurons of the rat. In colchicine-treated rats, a large number of Gal-immunoreactive cells were identified within all subdivisions of the tuberomammillary nucleus. The majority of these cells are large multipolar or fusiform neurons, with long, sparsely branching dendrites. A small number project to the ventral hippocampus, as shown by experiments with the retrograde tracing of Fast Blue. Ultrastructural examination of the Gal-immunoreactive cells confirms their indentity as magnocellular neurons, with dense deposits of immunoreaction product, particularly in small ribosomal arrays and in large, dense-cored vesicles. Axosomatic synapses occur on these neurons. The axonal boutons synapse with asymmetric and symmetric junctions and contain small synaptic vesicles as well as numerous large, dense-cored vesicles, which display Gal immunoreactivity. Sequential staining of thin, alternate sections with antibodies against Gal and L-histidine decarboxylase (HDCase; EC 4.1.1.22) showed colocalization of Galand HDCase-immunoreactivities in a majority of tuberomammilary neurons. The finding of Gal immunoreactivity within histamine-producing neurons of the tuberomammillary nucleus adds to the multiplicity of potential neuronal messengers utilized by these cells.

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of 13 formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by blot hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons.

The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods. The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery. In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells. In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.

The neuroendocrine marker neuron-specific enolase (NSE) has been reported to be detectable by immunohistochemistry in paraffin sections of neuroendocrine neoplasms of the lung. There is no study of immunodetection of NSE in cytological smear preparations from these tumours. We have examined 10 cases of small cell carcinoma of the lung, using cells obtained from serous fluids or bronchial biopsies, and found all but one had NSE-like immunoreactivity. No such immunoreactivity was found in 26 serous fluids and 19 biopsies from non-small-cell carcinomas. It is suggested, therefore, that immunostaining for NSE is a valuable aid to the cytological diagnosis of small cell carcinoma of the lung.

In human airways synthetic human sequence calcitonin gene-related peptide (hCGRP), a novel peptide produced by alternative processing of mRNA from the calcitonin gene, caused concentration-dependent contraction of human bronchi (EC50 4.9 X 10(-9) M) and was significantly more potent than substance P or carbachol. The contractile response was unaffected by atropine (2 X 10(-6) M), propranolol (10(-6) M), indomethacin (10(-5) M), tetrodotoxin (3 X 10(-6) M), chlorpheniramine (10(-4) M), cimetidine (10(-5) M), or FPL55712 (10(-4) M) suggesting a direct effect of CGRP on airways smooth muscle. CGRP was detected in human airways by radioimmunoassay with highest concentrations in cartilaginous airways. CGRP was localised by immunocytochemistry to both nerves and ganglia in human airways. CGRP, is a potent constrictor of human airways and may have important effects on airway function and be implicated in the pathogenesis of bronchial hyper-responsiveness and asthma.