Small intensely fluorescent (SIF) cells and sympathetic nerves in the adult rabbit portal vein and during perinatal development

Summary The ontogenesis of small intensely fluorescent (SIF) cells and the adrenergic nerve plexus is described in stretch preparations of the rabbit portal vein. On the 25 to 26th days of gestation there was a predominance of SIF cells (8 to 30 m in diameter), but a few nerve fibres in bundles were also present. Each portal vein preparation contained 6 to 9 groups of cells. The distribution and number of SIF cells and nerve bundles remained constant until the 31st day of gestation at which stage the number of SIF cells had decreased, while the density of the nerve plexus had increased approximately 4-fold. The adult portal vein exhibited a dense adrenergic plexus, but SIF cells were absent from nine out of ten preparations.     

Histofluorescence and ultrastructural observations of small intensely fluorescent (SIF) cells in the superior sympathetic ganglion of the guinea pig

Summary Amine-containing small intensely fluorescent (SIF) cells are ubiquitous in vertebrate sympathetic ganglia and, in some species, SIF cells have been identified as interneurons. The hypothesis proposed in this study is that SIF cells in superior sympathetic ganglia of the guinea pig function as interneurons, with efferent connections characteristic for the species. Fluorescence (catecholamine) microscopy and 5-hydroxydopamine marker for electron microscopy were used to study SIF cells, their processes and connections in this ganglion.Brightly fluorescent fibers were seen attached to virtually all SIF cells, and were of two types. The first type, single or arranged in cords, interconnected elements of the SIF-cell system; these apparent linkages joined individual SIF cells as well as adjacent clusters. The electron-microscopic evidence for synaptic contacts between SIF cells warrants the claim that integrated action is a presumed function of these elements. The second type of SIF-cell process was generally of greater length. These individual, branching fibers made presumed connections with dendrites of most principal ganglionic neurons. This arrangement suggested by histofluorescence preparations was confirmed by electron microscopy to involve synaptic connections, and the postsynaptic element was shown to be continuous with the perikaryon of the principal ganglionic neuron. Ultrastructural evidence that collections of dense-cored vesicles occur within processes of both principal ganglionic neurons and SIF cells, in proximity to unsheathed portions of plasma membrane, leads to the conclusion that interstitial diffusion of catecholamine from both may occur; the finding of SIF cell processes adjacent to fenestrated blood vessels suggests that catecholamine may also be transported through capillaries.     

Adrenergic and cholinergic nerves of bovine,guinea pig and hamster salivary glands

Summary The distribution of formaldehyde-induced fluorescence and acetylcholine-esterase (AChE) activity was histochemically investigated in certain salivary glands of the cow (submandibular gland), guinea pig and hamster (submandibular and sublingual glands). Adrenergic nerves occur around the secretory acini of the bovine, guinea pig and hamster submandibular glands, as well as around those of the hamster sublingual gland. The mucous secretory acini of the guinea pig sublingual gland, however, seem to be devoid of adrenergic nerve supply. Except in the sublingual gland of the hamster, no adrenergic nerves occur in relation to duct cells.The pattern of AChE activity is similar to that of adrenergic nerves. Thus, AChE-positive nerves form a network around secretory acini of all the five glands examined. Furthermore, AChE activity was also observed in nerve fibres in close proximity to striated duct cells.Both adrenergic and AChE-containing fibres were observed around blood vessels of different sizes. Ganglionic cells are occasionally to be seen; they all display AChE-activity. No adrenergic ganglionic cells were observed in any of the glands examined.All glands were also studied in the electron microscope. Interest was focussed on the fine structure of the autonomic nerves with special reference to their contents and type of storage vesicles.The content of noradrenaline was chemically determined in each type of salivary gland studied.This work was supported by grants from the University of Umeå and from the Swedish Society for Medical Research and was also carried out within a research organization supported by the Swedish Medical Research Council (projects B73-04X-712-08C and B73-04X-56-09C). The authors are indebted to Miss Kristina Karlsson and Miss Marianne Borg for valuable technical assistance.     

Intraganglionic portal sinus located between small intensely fluorescent (SIF) cells and principal ganglionic neurons in the inferior mesenteric ganglion of the guinea pig

Summary The vascular system in the inferior mesenteric ganglion of the guinea pig was studied to clarify the transport pathway of transmitters released by the small intensely fluorescent (SIF) cells to the principal ganglionic neurons. Reconstruction of about 1500 1-m-thick serial sections of the ganglion demonstrated its portal system. SIF cells were tightly packed and formed two or three clusters under the capsule of the ganglion. Branches from the inferior mesenteric artery ran directly toward these clusters and broke up into a number of coiled and looped sinusoid capillaries among the SIF cells. They then drained into a large sinus surrounding the clusters in the ganglion. Capillaries were derived from this sinus and ramified among the principal ganglionic neurons. After supplying the neurons, these vessels drained into veins surrounding the ganglion. Therefore, as we observed two distinct groups of capillaries, we call this sinus the intraganglionic portal sinus. All the transmitters secreted from the SIF cells are collected into this intraganglionic portal sinus and are then conveyed through the capillaries to the principal ganglionic neurons.     

Coexistence of multiple peptides in small intensely fluorescent (SIF) cells of inferior mesenteric ganglion of the guinea pig

Summary Coexistence of peptides in the small intensely fluorescent cells was demonstrated by immunocytochemistry for met-enkephalin-Arg-Gly-Leu, vasoactive intestinal polypeptide, somatostatin, neuropeptide Y and dynorphin. In the extreme example, a single cell was immunoreactive to all 5 peptides examined. Four peptides coexisted in 8% and three peptides in 13% of SIF cells. In 10% of SIF cells no peptide immunoreactivity could be detected. The most prevalent peptide was met-enkephalin (in 46% of cells), then vasoactive intestinal polypeptide (45%), somatostatin (39%), neuropeptide Y (31%) and dynorphin (24%). Met-enkephalin and vasoactive intestinal polypeptide coexisted most commonly (25%).     

Sympathetic and sensory innervation of small intensely fluorescent (SIF) cells in rat superior cervical ganglion

The sympathetic ganglion contains small intensely fluorescent (SIF) cells derived from the neural crest. We morphologically characterize SIF cells and focus on their relationship with ganglionic cells, preganglionic nerve fibers and sensory nerve endings. SIF cells stained intensely for tyrosine hydroxylase (TH), with a few cells also being immunoreactive for dopamine β-hydroxylase (DBH). Vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were distributed around some clusters of SIF cells, whereas some SIF cells closely abutted DBH-immunoreactive ganglionic cells. SIF cells contained bassoon-immunoreactive products beneath the cell membrane at the attachments and on opposite sites to the ganglionic cells. Ganglion neurons and SIF cells were immunoreactive to dopamine D2 receptors. Immunohistochemistry for P2X3 revealed ramified nerve endings with P2X3 immunoreactivity around SIF cells. Triple-labeling for P2X3, TH and VAChT allowed the classification of SIF cells into three types based on their innervation: (1) with only VAChT-immunoreactive puncta, (2) with only P2X3-immunoreactive nerve endings, (3) with both P2X3-immunoreactive nerve endings and VAChT-immunoreactive puncta. The results of retrograde tracing with fast blue dye indicated that most of these nerve endings originated from the petrosal ganglion. Thus, SIF cells in the superior cervical ganglion are innervated by preganglionic fibers and glossopharyngeal sensory nerve endings and can be classified into three types. SIF cells might modulate sympathetic activity in the superior cervical ganglion.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

Intramural neurons in the urinary bladder of the guinea-pig

Summary The urinary bladder of adult female guinea-pigs was stained histochemically to detect the presence of intramural ganglion neurons. Counts on wholemount preparations of entire bladders revealed the presence of 2000–2500 neurons per bladder, either as individual nerve cells or, more often, as ganglia containing up to 40 neurons. Both ganglia and single neurons lie along nerve trunks and are interconnected to form a plexus. Ganglia occur in every part of the bladder; they are more numerous on the dorsal than on the ventral wall, and they are especially abundant in an area within a radius of 800 m from the point of entry into the bladder wall of ureters and urinary arteries. The ganglia are located inside the muscle coat and close to muscle bundles; they usually lie nearer the mucosa than the serosa. Ultrastructurally, each ganglion is surrounded by a capsule; in addition to neurons and glial cells, the ganglia contain capillaries, collagen fibrils and fibroblasts; ganglion neurons are individually wrapped by glial cells and are separated from one another by connective tissue.     

A comparative and quantitative study of small intensely fluorescent (SIF) cells in the sympathetic ganglia of some small mammals

This comparative study of the number of SIF cells in the ganglions of the rat, cat, rabbit, mouse and hamster has confirmed that the mean number of SIF cells in the same ganglion of different species varies greatly, for instance in the superior cervical ganglion (SCG) of the rat and the cat, in the stellate ganglion of the cat and the mouse, or in the inferior mesenteric ganglion of the hamster and the other species. There is also considerable variability among individuals of the same animal species. In the SCG, the only ganglion for which there are data on the number of neurons, the ratio of SIF cells to neurons is around 1% in the rat, 0.2% in the rabbit, 0.3% in the mouse and 0.05% in the cat, i.e. a twenty-fold difference between the cat and the rat. Williams et al. (1975) distinguished type 1 SIF cells, corresponding to interneurons, from type 2, which are purely endocrine cells. Type 2 appears to be predominant in all ganglia, except the rabbit SCG where type 1 is highly predominant, and in all species, except the rat, in which this distinction is not applicable. The possible implications of these data on ganglionic functioning are discussed.     

The origin of VIP-containing nerves in the urinary bladder of rat

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.     

Effect of hydrocortisone on the ultrastructure of the small,intensely fluorescent,granule-containing cells in cultures of sympathetic ganglia of newborn rats

Summary Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with or without hydrocortisone. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde solution and thereafter in 1% osmium tetroxide. Hydrocortisone (10 mg/l) caused a great increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants, and the fluorescence intensity of these cells was also increased. The SIF cells corresponded to small, granule-containing (SGC) cells in the electronmicros copic preparations, and in addition to an increase in their number there was also an increase in the size and number of granular vesicles in the presence of hydrocortisone. In control cultures the granular vesicles were round (about 100 nm in diameter) or elongated (40–150 nm in cross section and 150–250 nm in length); both types of vesicles contained electron dense cores. In hydrocortisone-containing cultures round granular vesicles up to 200 nm in diameter were also observed; the cores of these vesicles were of variable electron density. It is concluded that in tissue culture, hydrocortisone causes an increased formation of catecholamine-containing granular vesicles in SIF-SGC cells and their precursors and an increase in the number of these cells.This work was supported by grants from the National Heart Foundation, the Australian Research Grants Committee and the Sigrid Juselius Foundation.University of Melbourne Senior Research Fellow, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.Holder of a grant from the National Health and Medical Research Council of Australia.Sunshine Foundation and Rowden White Research Fellow in the University of Melbourne, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.     

A time course study of water permeability and morphological alterations induced by mucosal hyperosmolarity in frog urinary bladder

Summary The role of the tight junction in the hydrosmotic response of the frog urinary bladder has been analysed by comparative kinetic studies and freeze etching examination. The comparison of the time course of the variations in transepithelial water net flux and of the alterations of tight junction ultrastructure in bladders exposed to mucosal hyperosmolar solutions shows that blisters are present in the tight junction before any increase in transepithelial water net flux. This indicates that the two phenomena are dissociated.In the same experimental conditions, freeze etching examination shows the presence in the tight junction of large areas of smooth and apparently stretched membrane where the typical network structure has disappeared. These alterations are reduced by further treatment with oxytocin and are probably not involved in the physiological hydrosomotic response.This work was supported in part by a grant from the Medical Research Council of Canada (J.H.).     

Principal neurons projecting to the pineal gland in close association with small intensely fluorescent cells in the superior cervical ganglion of rats

Summary The localization in the superior cervical ganglia (SCG) of small, intensely fluorescent (SIF) cells and of principal nerve (PN) cells innervating the pineal gland was examined in adult male Sprague-Dawley rats. PN cells were demonstrated by means of the retrograde neuron-tracing method using the fluorescent tracer Fluoro-Gold (FG) injected into the pineal gland. SIF cells were visualized by the formaldehyde-induced fluorescence method. Twentynine percent of the FG-labeled PN cells were found closely associated with SIF cells. In the rostral half of the ganglion, 43% of the SIF cells were situated in juxtaposition to one or several labeled neurons. The possible influence of SIF cells on the regulation of pineal metabolism is discussed with respect to their role as both local endocrine cells and interneurons.     

Origin and distribution of neuropeptide Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibers in the urinary bladder of the rat

Summary The origin and distribution in the urinary bladder of nerve fibers containing neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and substance P (SP) were investigated in rats. Experimental procedures comprised preganglionic decentralization or postganglionic denervation of the bladder and also chemical sympathectomy as well as capsaicin treatment of newborn rats.Nerve fibers containing NPY were richly distributed in the detrusor muscle and also in the pelvic ganglia. Numerous NPY-containing nerve cell bodies were found in pelvic ganglia. A rich occurrence of VIP fibers and a more sparse distribution of SP-containing fibers were also found in the bladder as well as a relatively rich representation of VIP- containing nerve cell bodies in the pelvic ganglia. After decentralization the intensity of VIP and NPY immunofluorescence increased in nerve cell bodies of the pelvic ganglia and in nerve fibers in the wall of the bladder. Postganglionic denervation, on the other hand, eliminated all peptides examined in the bladder wall. After postganglionic denervation the situation in the ganglia was approximately the same as after decentralization. Chemical sympathectomy (6-OHDA) did not seem to change significantly the frequency and distribution of VIP-, SP- and NPY-fibers in the muscle layer of the bladder or in the pelvic ganglia, while the NPY-containing nerve fibers in the submucosal layer and around blood vessels of the bladder disappeared. Adrenergic nerve fibers in the wall of the bladder (visualized by histofluorescence) were markedly reduced in number after administration of 6-OHDA. Capsaicin-treatment of newborn rats caused a loss of SP-fibers in the wall of the bladder, supporting the view that these fibers are sensory in nature in the urinary bladder. Although it cannot be entirely excluded that NPY-containing fibers in the wall of the bladder are adrenergic, the present results suggest that the NPY-fibers as well as the VIP-fibers of the bladder wall originate mainly in non-adrenergic cell bodies of the pelvic ganglia. However, perivascular NPY-containing nerve fibers are adrenergic in nature.     

Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

The effect of 17-B-estradiol on the fine structure of the adrenergic axons of the rabbit myometrium

Summary The effects of 17-B-estradiol on the fine structure of the autonomic nerves of the rabbit oviduct and myometrium were studied by means of KMnO₄-fixation. The main changes due to prolonged estrogen treatment were the following: (1) the dimensions of the axons increased, (2) the amount of smooth endoplasmic reticulum in the axons increased, (3) the amounts of both large granular vesicles (LGV) and large agranular vesicles (LAV) in the axons increased and (4) electron dense grains were found within the endoplasmic tubuli of the axons. Furthermore, it seems probable that the amount of small granular vesicles was also considerably higher after two weeks of estrogen treatment.The mechanism of estrogen action on the storage of transmitter within the axon is discussed. In conclusion, the present results suggest that both the axoplasmic transport and the peripheral formation of storage sites for noradrenaline are stimulated by the increased estrogen level.This work was supported by grants from Orion, Helsinki and from Finska Läkaresällskapet.