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.     

Relationship of blood vessels to small intensely fluorescent cells in the autonomic ganglion.

In our experiments we observed the relationship of the blood vessels to the small, intensely fluorescent cells (SIF cells) in the lower mesenteric ganglion of the cat. We injected the solution of Evan's blue into the ganglia and processed them with the fluorescent histochemical method by Falck and Hillarp. We observed that the SIF cells are placed in the ganglia closely to the blood vessels or closely round them. When observing lager groups of SIF cells placed at the edge of the ganglia a dense network of the blood vessels was observed among these cells.     

Cytochrome b 558 and hydrogen peroxide production in small intensely fluorescent cells of sympathetic ganglia

 Small intensely fluorescent (SIF) cells are paraganglionic cells derived from sympathicoblasts which may serve as interneurons, endo-/paracrine cells or arterial chemoreceptors within sympathetic ganglia. Like paraganglionic cells of other locations, e.g., carotid body glomus cells, they are responsive to hypoxia. Recent studies on glomus cells and other hypoxia-sensing cells suggested the involvement of a b ₅₅₈ -type cytochrome and intracellular generation of H₂O₂ in the process of oxygen sensing. In the present study, we demonstrate the occurrence of the small subunit of cytochrome b ₅₅₈ , p22phox, in SIF cells of guinea-pig sympathetic ganglia by immunohistochemistry using two different antisera. H₂O₂ production was monitored in explanted intact superior cervical ganglia of 2-day-old rats by confocal laser scanning analysis of rhodamine 123 fluorescence generated due to oxidation of dihydrorhodamine 123 by H₂O₂. Using this technique, SIF cell clusters appeared as sites of highest H₂O₂ production within the ganglia. Thus, SIF cells exhibit two key features of an oxidase system generating reactive oxygen species. This may be involved in the proposed chain of events in oxygen sensing, but alternative cellular functions of this system have also to be considered. Accepted: 19 September 1996     

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.     

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.     

Differentiation of noradrenergic traits in the principal neurons and small intensely fluorescent cells of the parasympathetic sphenopalatine ganglion of the rat

Catecholamine synthetic enzymes are found in many cranial parasympathetic principal neurons, and in the small intensely fluorescent (SIF) cells that populate parasympathetic as well as sympathetic ganglia. While there is evidence that the acquisition of noradrenergic properties in sympathetic neuron precursors depends on factors that these cells encounter in the trunk environment, the mechanisms that direct the development of noradrenergic traits in cranial parasympathetic neurons and SIF cells are not understood. The present study examines the time course of appearance of tyrosine hydroxylase (TH) immunoreactivity in the principal neurons and SIF cells of the rat sphenopalatine ganglion. We show that the sphenopalatine ganglion of normal adult rats contains both a small population of TH-immunoreactive principal neurons and many SIF cells. The TH-immunoreactive principal neurons do not synthesize or store detectable catecholamines, even though the majority of sphenopalatine ganglion neurons do contain 1-amino acid decarboxylase catalytic activity. Sphenopalatine ganglion principal neurons do not accumulate detectable levels of exogenous catecholamines. This observation suggests that they lack a high affinity norepinephrine uptake system. In contrast to what has been observed previously for sympathetic neurons, the appearance of TH immunoreactivity in sphenopalatine neurons is not temporally correlated with the cessation of neural crest cell migration. The first TH-immunoreactive neurons do not appear in the sphenopalatine ganglion until Embryonic Day 16.5, 2 days after the ganglion has condensed and process outgrowth has begun. The number of sphenopalatine neurons that express TH immunoreactivity increases dramatically between Embryonic Day 18.5 and Postnatal Day 1, but then decreases. In fact, the percentage of sphenopalatine neurons that express TH immunoreactivity is almost fivefold higher in newborn than in adult rats. SIF cells cannot be definitively identified in the sphenopalatine ganglion until after Embryonic Day 18.5. The time course of appearance of TH immunoreactivity in sphenopalatine ganglion cells raises the possibility that TH expression is stimulated in these cells by factors encountered either at their condensation site or at their target, such as glucocorticoids or nerve growth factor. The relatively late appearance of SIF cells in the sphenopalatine ganglion argues against the hypothesis that SIF cells are the precursors of all autonomic 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%).     

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.     

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.     

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.     

Hydrocortisone-induced increase in the number of small intensely fluorescent cells and their histochemically demonstrable catecholamine content in cultures of sympathetic ganglia of the newborn rat

Synopsis It is known that hydrocortisone causes a great increase in the number of small intensely fluorescent (SIF) cells in the sympathetic ganglia when injected into newborn rats. The effect of hydrocortisone on nervous tissuein vitro has not been studied previously.Pieces of newborn rat sympathetic ganglia were cultivated in Rose chambers. Hydrocortisone was dissolved in the medium in concentrations of 1–9 mg/l. Both control and hydrocortisone-containing cultures were examined daily by phase-contrast microscopy, and the catecholamines were demonstrated histochemically by formaldehyde-induced fluorescence after 7 days in culture.All cultures showed outgrowths of axons and supporting cells elements, although these were less extensive in the groups of cultures with hydrocortisone. After a week, SIF cells with a green fluorescence were observed in the control explants. In all cultures with hydrocortisone, a concentration-dependent increase was observed in the fluorescence intensity and the number of the SIF cells in the explant; numerous SIF cells were also seen in the outgrowth. Some SIF cells showed processes and the longest processes were seen in cultures with the highest concentration of hydrocortisone.It is concluded that hydrocortisone causes an increased synthesis of catecholamines in the SIF cellsin vitro, and an increase in their number by affecting either their division or their differentiation from a more immature form, or both. This effect was a direct one and not mediated by any system other than the ganglion itself. Induction of enzyme synthesis by hydrocortisone is proposed as an explanation of the increase in catecholamine concentration.University of Melbourne Senior Research Fellow, September 1971-August 1972Sunshine Foundation and Rowden White Trust Overseas Research Fellow in the University on Melbourne, September 1971-August 1972     

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.     

The distribution of noradrenergic nerves and small,intensely fluorescent (SIF) cells in the cat urinary bladder

Summary Fluorescence and electron microscopy have been used to study the distribution of noradrenergic nerves in the smooth muscle of the cat urinary bladder. Using the former technique, relatively few fluorescent noradrenergic nerves were observed in the body and fundus, while a rich plexus occurred adjacent to muscle cells of the bladder neck. The trigone could not be distinguished neuromorphologically from detrusor muscle in this region. Electron microscopy showed that the majority of noradrenergic terminals in the body and fundus were associated with presumptive cholinergic axons, while in the bladder neck noradrenergic terminals formed typical neuroeffector relationships with individual smooth muscle cells.Numerous ganglia occurred both in the adventitia and among the smooth muscle bundles, particularly in the bladder neck. The majority of the nerve cell bodies were non-fluorescent, although many contained bright orange autofluorescent granules, believed to be lysosomes. A small minority of ganglion cells were associated with fluorescent noradrenergic nerve terminals, thereby providing structural evidence for limited intraganglionic inhibition. In addition, occasional groups of small intensely fluorescent (SIF) cells were observed in some intramural ganglia and these were subsequently identified in the electron microscope. The possibility that these cells may provide a second inhibitory influence on bladder activity was considered.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Calcium and action potentials of bullfrog sympathetic ganglion cells

Bullfrog sympathetic ganglion cells were capable of producing action potentials (Ca spikes) in an isotonic (84 mM) CaCl2 solution. The peak level of Ca spikes showed an approximately 30 mv increase with a 10-fold increase in the Ca concentration. Na as well as Ca ions were capable of acting as charge carriers during the production of action potentials in a solution containing relatively high Ca and relatively low Na ions. A decrease in the external Ca concentration depressed the maximum rate of rise at a fixed resting potential level, and increased the maximum rate of rise of the Na spikes at a high resting potential level at which Na inactivation was completely depressed. Compared to Na spikes, Ca spikes were less sensitive to TTX and procaine. Ganglion cells were also capable of producing action potentials (Sr spikes) in an isotonic SrCl₂ solution and prolonged action potentials in an isotonic BaCl₂ solution, but these cells were rendered inexcitable in an isotonic MgCl₂ solution. The peak level of the Sr spikes was dependent on the external Sr concentration and was insensitive to both TTX and procaine. Sr ions, like Ca ions, reduced Na inactivation during the resting state, and depressed the maximum rate of rise of the Na spikes at a high resting potential level. It was concluded that Ca (and Sr) ions exert dual actions on the membrane; namely, regulating the Na permeability and acting as charge carriers during the active state of the membrane.