Small Intensely Fluorescent Cells Research Articles

Expression of the gap junction protein connexin36 in small intensely fluorescent (SIF) cells in cardiac parasympathetic ganglia of rodents

In mammals, several endocrine cell types are electrically coupled by connexin36 (Cx36)-containing gap junctions, which mediate intercellular communication and allow regulated and synchronized cellular activity through exchange of ions and small metabolites via formation of intercellular channels that link plasma membranes of apposing cells. One cell type thought to be endocrine-like in nature are small intensely fluorescent (SIF) cells that store catecholamines in their dense-core vesicles and reside in autonomic ganglia. Here, using immunofluorescence approaches, we examined whether SIF cells located specifically in cardiac parasympathetic ganglia of adult and neonatal mice and adult rats follow patterns of Cx36 expression seen in other endocrine cells. In these ganglia, SIF cells were identified by their distinct small soma size, autofluorescence at 475 nm, and immunolabelling for their markers tyrosine hydroxylase and vesicular monoamine transporter-1. SIF cells were often found in pairs or clusters among principal cholinergic neurons. Immunofluorescence labelling of Cx36 occurred exclusively as fine puncta that appeared at contacts between SIF cell processes and somata or at somato-somatic appositions of SIF cells. These puncta were absent in cardiac parasympathetic ganglia of Cx36 null mice. Transgenic mice expressing enhanced green fluorescent protein reporter for Cx36 expression displayed labelling for the reporter in SIF cells. The results suggest that Cx36-containing gap junctions electrically couple SIF cells, which is consistent with previous suggestions that these may be classified as endocrine-type cells that secrete catecholamines into the bloodstream in a regulated manner.

Identification of glucocorticoid receptors as potential modulators of parasympathetic and sympathetic neurons within rat intracardiac ganglia.

BackgroundEmerging evidences indicate that glucocorticoid receptors (GR) play a regulatory role in cardiac function, particularly with regard to the autonomic nervous system. Therefore, this study aimed to demonstrate the expression and the precise anatomical location of GR in relation to the parasympathetic and sympathetic innervations of the heart.MethodsThe present study used tissue samples from rat heart atria to perform conventional reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and double immunofluorescence confocal analysis of GR with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) as well as the mineralocorticoid receptor (MR).ResultsDouble immunofluorescence labeling revealed that GRs were co-expressed with VAChT in parasympathetic principal neuronal somata and nerve terminals innervating atrium. Also, GR colocalized with the sympathetic neuronal marker TH in a cluster of small intensely fluorescent (SIF) cells, on intracardiac nerve terminals and in the atrial myocardium. GR immunoreactivity was scarcely identified on CGRP-immunoreactive sensory nerve terminals. Approximately 20% of GR immunoreactive neuronal somata co-localized with MR. Finally, conventional RT-PCR and Western blot confirmed the presence of GR and MR in rat heart atria.ConclusionThis study provides evidence for the existence of GR predominantly on cardiac parasympathetic neurons and TH-immunoreactive SIF cells suggesting a functional role of cardiac GR on cardiovascular function by modulation of the cardiac autonomic nervous system.

Immunohistochemical localization of vesicular nucleotide transporter in small intensely fluorescent (SIF) cells of the rat superior cervical ganglion

Our previous study reported that a part of small intensely fluorescent (SIF) cells in the rat superior cervical ganglion were innervated by P2X3 purinoceptor-expressing glossopharyngeal sensory nerve endings, suggesting the occurrence of adenosine 5′-triphosphate (ATP)-mediated transmission between them. The present study investigated the immunolocalization of vesicular nucleotide transporter (VNUT) in SIF cells of the superior cervical ganglion in male Wistar rats. VNUT was immunohistochemically localized in tyrosine hydroxylase-immunoreactive SIF cells and sympathetic postganglionic neurons, but not in a few SIF cells with immunoreactivity for dopamine beta-hydroxylase. P2X3-immunoreactive ramified nerve endings formed flat leaf-like or spherical terminal parts to surround some VNUT-immunoreactive SIF cells, but not other VNUT-immunoreactive SIF cells attached to ganglionic neurons. VNUT-immunoreactive SIF cells contained bassoon-immunoreactive products at the contacting surface of P2X3-immunoreactive nerve endings. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2, which hydrolyzes extracellular ATP, was observed in S100B-immunoreactive satellite glial cells surrounding VNUT-immunoreactive SIF cells, but not in the attachment surfaces between SIF cells and nerve endings with P2X3 immunoreactivity. The present results suggest that SIF cells release ATP by exocytosis to modulate the excitability of sensory nerve endings and postganglionic neurons in the superior cervical ganglion.

Topographical Mapping of Catecholaminergic Axon Distribution and Morphology in the Flat‐Mounts of the Mouse Heart

The sympathetic nervous system plays a crucial role in the control of heart functions. However, the distribution and morphology of sympathetic postganglionic innervation of the heart has not been well elucidated, which has significantly impeded the understanding of the sympathetic neuroanatomical organization and control of the heart. Our goal is to provide a map of the topographical innervation of sympathetic postganglionic neurons in the whole heart of mice. In this study, we used Tyrosine hydroxylase (TH) as a marker for sympathetic axons and applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing and immunohistochemistry, Neurolucida 360 Tracing and integration of the tracing data onto a 3D heart scaffold. We found that 1) Multiple large extrinsic TH‐IR nerve bundles entered the heart and branched out into smaller axons which covered the whole left and right atria and ventricles, 2) These TH‐IR bundles branched out into smaller axon bundles and ramified into individual axons covering the whole left and right atria and ventricles. 3) The TH‐IR axons formed an extensive neural network in the epicardium and axons formed varicose synaptic endings within the cardiac muscle. 4) TH‐IR axons and terminals were densest in the sinoatrial (SA) and atrioventricular (AV) node regions. 5) TH‐IR axons innervated blood vessels and fat cells. 6) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent (SIF) cells were TH‐IR. 7) However, TH‐IR axons do not appear to innervate any cardiac principal neurons and SIF cells. 8) TH‐IR axon innervation will be integrated into a 3D scaffold. Our work will provide a comprehensive topographical map of the catecholaminergic efferent axon distribution, innervation, and morphology of the whole heart at single cell/axon/synapse scale that will be used to create a cardiac sympathetic‐brain atlas. It will also provide an anatomical reference to evaluate the altered sympathetic cardiac control in pathological conditions. The first two authors contributed equally to this work.

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.

Developmental expression of δ‐opioid receptors during maturation of the parasympathetic, sympathetic, and sensory innervations of the neonatal heart: Early targets for opioid regulation of autonomic control

Evidence is accumulating regarding the local opioid regulation of heart function. However, the exact anatomical location of δ-opioid receptors (DORs) and expression during maturation of the autonomic and sensory innervations of the neonatal heart is unknown. Therefore, we aimed to characterize target sites for opioids in neonatal rat heart intracardiac ganglia at postnatal day (P)1, P7 and adulthood (P56-P84). Rat heart atria were subjected to reverse-transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis of DORs with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and substance P (SP). Our results demonstrated DOR mRNA, protein, and binding sites that gradually increased from P1 toward adulthood. Immunofluorescence confocal microscopy showed DOR co-localized with VAChT in large-diameter principal neurons, TH-immunoreactive (IR) small intensely fluorescent (SIF) catecholaminergic cells, and CGRP- or SP-IR afferent nerve terminals arborizing within intracardiac ganglia and atrial myocardium. Co-expression of DOR with VAChT-IR neurons was observed from the first day of birth (P1). In contrast, DORs on TH-IR SIF cells or CGRP-IR fibers were not observed in intracardiac ganglia of P1, but rather in P7 rats. The density of nerve fibers in atrial myocardium co-expressing DORs with different neuronal markers increased from neonatal age toward adulthood. In summary, the enhanced DOR expression parallel to the maturation of cardiac parasympathetic, sympathetic, and sensory innervation of the heart suggests that the cardiac opioid system is an important regulator of neonatal and adult heart function through the autonomic nervous system.

Distribution and fate of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp)‐expressing cells in rat urinary bladder: A developmental study

We examined the distribution and fate of cocaine- and amphetamine-regulated transcript peptide (CARTp)(55-102)-immunoreactive (IR) structures in the neonatal and adult rat urinary bladder. Double-labeling studies examining CARTp with tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), or choline acetyltransferase (ChAT) were performed in wholemounts of urothelium or detrusor or cryostat sections of the bladder. In younger animals (postnatal day [P]1, P3), CARTp-IR cell bodies in detrusor smooth muscle were observed in large clusters ( approximately 100 cells/cluster) at the ureteral insertion and along thick bundles of nerve fibers at the bladder base. The total number of CARTp-IR cells was significantly reduced (by five-fold) at P14, and this reduced number persisted into adulthood. The decrease in the number of CARTp-expressing cells was complemented with positive staining for cleaved caspase-3, suggesting that apoptosis contributed to this decrease. At birth (P1), all CARTp-IR cells expressed the neuronal marker Hu. After birth, CARTp was expressed by some neurons (CARTp-IR, Hu-IR) that represent intramural ganglion cells and by cells that lacked a neuronal phenotype (CARTp-IR, Hu-) but did express TH. Neither of these cell populations expressed ChAT immunoreactivity in adult bladder. These cells (CARTp-IR, Hu-, TH-IR) may represent paraganglion or small intensely fluorescent (SIF) cells. The percentage of colocalization of CARTp-IR and nNOS or TH was dependent on postnatal age and showed an inverse relationship. At P1, 67.1 % of CARTp-IR cells expressed nNOS immunoreactivity. Decreased colocalization was observed with increasing postnatal age. In contrast, 19.5% of CARTp-IR cells expressed TH at P1, but colocalization increased with postnatal age. The suburothelial plexus lacked CARTp-IR nerve fibers until P14, when nerve fibers with varicosities were observed in the urethra and bladder neck region. In summary, we demonstrate 1) a decrease in the number of CARTp-IR cells in rat detrusor in early postnatal development; 2) apoptotic events in the bladder during early postnatal development; 3) rostral migration of CARTp-IR cells from the ureteral insertion toward the bladder body during postnatal development; 4) the presence of different populations of CARTp-IR cells, some with and others without a neuronal phenotype; and (5) age-dependent changes in chemical coding of CARTp-IR cells with postnatal development. This study demonstrates that CARTp-IR intramural ganglia and CARTp-IR paraganglion or SIF cells exist in the postnatal and adult rat bladder, although the role of these cell types remains to be determined.

Catecholaminergic neurons in the rat intrinsic cardiac nervous system.

Immunoreactivities (IR) for catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DbetaH), phenylethanolamine N-methyl transferase (PNMT), serotonin-synthesizing enzyme tryptophan hydroxylase, and neuropeptide Y were investigated in the intrinsic cardiac nervous system of 27-40-day-old rats using fluorescent immunohistochemistry. Individual neurons were identified by the general neuronal marker protein gene product 9.5. The presence of DbetaH and PNMT in the atrial specimens was verified using reverse transcriptase-polymerase chain reaction. Two types of catecholamine-handling intrinsic ganglion neurons were observed: small intensely fluorescent (SIF) cells and large-diameter neurons. SIF cells exhibited TH- and tryptophan hydroxylase-IR, but they were not positive for DbetaH. In contrast, large-diameter intrinsic TH-positive neurons, showing in majority also NPY-IR, displayed also DbetaH- and PNMT-IR, thus indicating the capacity for the synthesis of norepinephrine and epinephrine, respectively. In conclusion, the SIF cells are most probably dopaminergic and serotonergic neurons, whereas large-diameter intrinsic cells seem to represent a subpopulation of norepinephrine- and/or epinephrine-secreting neurons.

The development of synaptophysin immunoreactivity in the human sympathetic ganglia

Using an indirect immunohistochemical method, synaptophysin immunoreactivity (SYN-IR) has been studied in cryostat sections of stellate and thoracic ganglia in human fetuses, neonates, infants and adults. In the course of development, a progressive increase in SYN-IR in axonal terminals and around nerve cells was demonstrated. In contrast, large clusters of small intensely fluorescent (SIF) cells and paraganglionic cells increased in number in fetuses and premature neonates at 24-25 weeks. Such SIF cell clusters varied in form and often occurred at pole or subcapsular areas of sympathetic ganglia close to blood vessels or paraganglia. With increasing gestational age and during infancy, a decrease in sizes of SIF cell groups and paraganglionic cells as well as changes in their distribution were found. The results show that the amount and distribution of SYN-IR is temporally related to the maturation and functional activity of human sympathetic ganglia neurons. It was suggested that numerous SIF cells and paraganglia in human prenatal sympathetic ganglia were both indicative of incomplete cell migration and an important source of regulation of ganglionic microcirculation under the conditions of relative hypoxia and immature nervous regulation.

Molecular cues for the development of adrenal chromaffin cells and their preganglionic innervation.

Based on recent evidence from in vitro and gene knockout/insertion studies, this short review summarizes the molecular scenario underlying the development of adrenal chromaffin cells and their preganglionic innervation. During migration of neural crest cells from the dorsal surface of the neural tube to their destinations in the sympathetic primordia and adrenal glands, precursors of the so-called sympathoadrenal (SA) cell lineage are exposed to signals from the notochord and ventral neural tube probably including the protein, Sonic hedgehog. These, and signals in the region of the dorsal aorta (members of the family of bone morphogentic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurones, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells, glucocortiocids have been claimed as essential for suppressing neuronal commitment and for channelling SA cells towards the chromaffin phenotype. However, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth, suggesting that signals other than glucocorticoid hormones may be important in triggering chromaffin cell differentiation. The cholinergic neurones that are preganglionic to adrenal chromaffin cells have their cell bodies located in the intermediolateral column (IML) of the spinal cord. For their normal development, these neurones require signals from the adrenal medulla, which include neurotrophin-4, a major neurotrophic factor of adrenal chromaffin cells. Taken together, these data provide a more complete picture of molecular signalling in the development of one of the most important neuroendocrine tissues in vertebrates.

Small intensely fluorescent cells of the rat paracervical ganglion synthesize adrenaline, receive afferent innervation from postganglionic cholinergic neurones, and contain muscarinic receptors

In the paracervical ganglion (PCG) of the rat, double-labelling immunofluorescence for catecholamine-synthesizing enzymes and HPLC measurement of catecholamine contents were first performed to evaluate whether intraganglionic small intensely fluorescent (SIF) cells are capable of synthesizing adrenaline. Immunolabelling for tyrosine hydroxylase (TH), dopamine β-hydroxylase and phenylethanolamine- N-methyl transferase (PNMT) occurred in all SIF cells of the PCG, thus demonstrating the presence of all the enzymes required for adrenaline biosynthesis. Adrenaline levels were undetectable in the PCG but to test the hypothesis that PNMT is active in SIF cells, catecholamines were measured in ganglia of rats pretreated with pargyline, an inhibitor of the monoamine oxidase, the major enzyme involved in the catecholamine degradation. Pargyline treatment increased adrenaline levels in the PCG, thus demonstrating that SIF cells are capable of adrenaline synthesis. The undetectable levels of adrenaline in the PCG of untreated rats suggested a slow rate of biosynthesis of adrenaline in the ganglion. Furthermore, the use of double-labelling showed that SIF cells of the PCG were stained for muscarinic receptors and were approached by varicose ChAT-immunoreactive nerve fibres. Nerve fibres immunoreactive for ChAT were also observed associated with nerve cell bodies of ganglion neurones. Following deafferentation of the PCG, the ChAT-immunoreactive nerve fibres surrounding nerve cell bodies totally disappeared indicating their preganglionic origin, while those associated with SIF cells did not degenerate, which demonstrate that they derived from intraganglionic cholinergic neurones. Taken together, the results show that adrenaline may be a transmitter for SIF cells in the PCG and suggest that cholinergic neurones of the parasympathetic division of the PCG can modulate the SIF cell activity through the activation of muscarinic receptors.

Vesicular amine transporter expression and isoform selection in developing brain, peripheral nervous system and gut

The vesicular monoamine transporters VMAT1 and VMAT2 are essential components of monoaminergic neurons and endocrine cells whose expression in development may provide insight into lineage pathways for chemical coding in the diffuse neuroendocrine system. Thus, the brain is a compartment in which only monoaminergic neurons are generated, the gut epithelium generates only endocrine monoamine-containing cells, and the neural crest produces both autonomic monoaminergic neurons and endocrine/paracrine monoaminergic cells. Selection of either the VMAT1 or VMAT2 isoform was examined in these three compartments during development. In the central nervous system VMAT2, but not VMAT1, was expressed in neuroepithelial cells by embryonic day 12 (E12), and all major monoaminergic cell groups by E14. Thalamocortical and hypothalamic neurons that do not express VMAT2 in adulthood were transiently VMAT2-positive from E16 to postnatal day 6 (P6). EC cells of the gut expressed exclusively VMAT1 from E19 on, while histamine-containing enterochromaffin-like (ECL) cells of the stomach expressed only VMAT2 by E19 and throughout postnatal development. VMAT2 and the vesicular acetylcholine transporter VAChT were co-expressed in early development of the primary sympathetic chain as well as in the cranial parasympathetic ganglia. VAChT was progressively restricted to a small population of VMAT2-negative post-ganglionic neurons in the adult sympathetic chain, while VMAT2 expression persisted in sympathetic principal ganglion and SIF cells but was eventually extinguished in cranial parasympathetic ganglia. VMAT1 was co-expressed with VAChT and VMAT2 mRNA in the primary sympathetic chain on E12, but progressively restricted to small intensely fluorescent (SIF) and chromaffin cells thereafter. Thus, expression of the vesicular amine transporters appropriate for chemical coding of brain neurons and gut endocrine cells are pre-determined developmentally. In contrast, the neural crest-derived sympathoadrenal and neural crest-derived parasympathetic cell groups examined here initially co-express two or more vesicular amine transporters, followed by extinction of the inappropriate transporter(s) later in development. Some neural crest-derived neuroendocrine cell populations continue to express both isoforms of VMAT even in adulthood. Lineage distinctions in ontogeny of vesicular amine transporter expression in brain, gut and autonomic nervous system make it likely that the same genes are regulated differently in the autonomic nervous system compared to brain and gut.

A laser confocal microscopic study of vagal afferent innervation of rat aortic arch: Chemoreceptors as well as baroreceptors

Although the aortic nerves contain vagal afferents that terminate in both the wall of the aortic arch (putative baroreceptors) and its associated glomus tissue (putative chemoreceptors) in most mammalian species, the aortic nerves of the rat have been widely assumed to contain only baro- or pressor afferents. The present study reconsidered this anomaly by characterizing vagal afferent endings and their targets in the aortic arch region of the rat, both qualitatively and quantitatively. Eight Sprague–Dawley rats received intracranial vagal motor rhizotomy unilaterally to eliminate efferents in the nerve and then, two weeks later, injections of the tracer DiI (1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate) into the ipsilateral nodose ganglion. The aortic arch and its surrounding tissue, with the common carotid and subclavian arteries attached, were examined with both conventional epifluorescence and confocal microscopes. Consistent with earlier observations, vagal afferents formed both flower-spray and end-net terminals rather diffusely within the wall of the aortic arch. More interestingly, vagal afferents also innervated glomus or SIF (i.e., small intensely fluorescent) cell bodies at the junction areas of the common carotid and subclavian arteries. To identify the course of these fibers, six additional animals received DiI injection into the nodose unilaterally after a complete cervical vagotomy caudal to the nodose; in these animals, the aortic nerve had been separated from the vagal trunk and kept intact. There were no marked differences in innervation patterns between the nonvagotomized and the cervically vagotomized animals, indicating that the vagal axons innervating the walls of the blood vessels and the SIF cells in the aortic arch region travel through the aortic nerves. Using a stereological method, we estimated the relative number of chemo- and baroreceptor afferents innervating the aortic arch. About 16.4% (left) and 13.1% (right) of fibers in the aortic nerves innervate SIF cells. These findings challenge the general consensus that the aortic nerves of rats contain exclusively baroreceptor fibers.

Generation of cell diversity in the peripheral autonomic nervous system: The sympathoadrenal cell lineage revisited

Based on recent evidence from in vitro and gene knock-out/knock-in studies this short review summarizes the molecular scenario underlying the development of autonomic neurons from the neural crest. The focus is on the sympathoadrenal (SA) cell lineage. While migrating ventrally precursors of this cell lineage are exposed to signals from notochord/ventral neural tube probably including the protein sonic hedgehog. These and signals in the region of the dorsal aorta (members of the family of bone morphogenetic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurons, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells glucocorticoids have been claimed as essential for suppressing a neuronal commitment and channeling SA cells towards the chromaffin phenotype. Unexpectedly, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth. We present a hypothetical scenario consistent with these data, in which chromaffin cell development would be the default pathway in the SA cell lineage, while development into a neuronal direction requires specific growth factor signaling, which is probably distinct for paravertebral and prevertebral sympathetic neurons.

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy.

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed "flower-spray" and "end-net" terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes.

Cytochrome b558 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 b558-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 b558, 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.

Muscarinic transmission decreases the number of SIF cells demonstrating catecholamine histofluorescence in rat superior cervical ganglia

Preganglionic electrical stimulation of the cervical sympathetic trunk to the rat superior cervical ganglia produced a mean reduction in the number of visible small intensely fluorescent (SIF) cells demonstrating catecholamine histofluorescence to 32% of the unstimulated contralateral control. The reduction in the number of catecholamine-positive SIF cells required the presence of specific blockers of catecholamine uptake and synthesis and was dependent on normal synaptic transmission. No change in the number of catecholamine-positive SIF cells was observed when ganglionic transmission occurred in solutions containing both hexamethonium and atropine or with atropine alone (97% of the unstimulated control). Furthermore, preganglionic stimulation in the presence of high magnesium/low calcium solutions, which effectively blocked synaptic transmission, prevented the stimulation-induced decrease in the number of catecholamine-positive SIF cells. Prolonged antidromic stimulation of the internal carotid nerve only reduced the number of catecholamine-positive SIF cells to 75% of the unstimulated contralateral control. These results suggest that preganglionic synaptic impulses can induce the release of catecholamines from SIF cells via muscarinic receptor activation. Furthermore, the necessity for pharmacological intervention of uptake and synthesis blockers of catecholamines in order to detect the synaptically-induced reduction in the number of catecholamine-positive SIF cells, suggests that synaptic transmission also modulates the synthesis of catecholamines in SIF cells within the rat superior cervical ganglia.

Microvascular organization of sympathetic ganglia, with special reference to small intensely-fluorescent cells.

The vascular organization of sympathetic ganglia has been reviewed in relation to type II small intensely-fluorescent (SIF) cells. These cells are considered to be secretory cells forming large clusters surrounded by fenestrated capillaries. Immunohistochemical studies have revealed the existence of many kinds of peptides, in addition to catecholamines, in type II SIF cells. These transmitters are thought to enter the bloodstream, perfuse the adjacent ganglionic tissue, and modify the synaptic transmission and activity of sympathetic ganglionic neurons. Several authors reported portal-like intraganglionic microcirculation through which type II SIF cells participate in modulation of the principal ganglionic neurons. One large intraganglionic portal sinus located between SIF cells and principal ganglionic neurons was also reported in the inferior mesenteric ganglion. However, some authors claimed that transmitters could be absorbed through numerous capillary anastomoses, without any portal system in the superior cervical ganglion. It is noticed that the number, size, and partition of SIF-cell clusters are variable in different ganglia and different animal species. It is important to interpret the functional and morphological correlates of intraganglionic microcirculation based on the species and location of ganglia.

Muscarinic transmission decreases the number of SIF cells demonstrating catecholamine histofluorescence in rat superior cervical ganglia

Preganglionic electrical stimulation of the cervical sympathetic trunk to the rat superior cervical ganglia produced a mean reduction in the number of visible small intensely fluorescent (SIF) cells demonstrating catecholamine histofluorescence to 32% of the unstimulated contralateral control. The reduction in the number of catecholamine-positive SIF cells required the presence of specific blockers of catecholamine uptake and synthesis and was dependent on normal synaptic transmission. No change in the number of catecholamine-positive SIF cells was observed when ganglionic transmission occurred in solutions containing both hexamethonium and atropine or with atropine alone (97% of the unstimulated control). Furthermore, preganglionic stimulation in the presence of high magnesium/low calcium solutions, which effectively blocked synaptic transmission, prevented the stimulation-induced decrease in the number of catecholamine-positive SIF cells. Prolonged antidromic stimulation of the internal carotid nerve only reduced the number of catecholamine-positive SIF cells to 75% of the unstimulated contralateral control. These results suggest that preganglionic synaptic impulses can induce the release of catecholamines from SIF cells via muscarinic receptor activation. Furthermore, the necessity for pharmacological intervention of uptake and synthesis blockers of catecholamines in order to detect the synaptically-induced reduction in the number of catecholamine-positive SIF cells, suggests that synaptic transmission also modulates the synthesis of catecholamines in SIF cells within the rat superior cervical ganglia.

Localization and axotomy-induced regulation of the peptide secretoneurin in the rat superior cervical ganglion.

This study demonstrates the localization and regulation of a novel neuropeptide of 33 amino acids, secretoneurin (SN), in the rat superior cervical ganglion. Gel filtration chromatography of ganglion proteins followed by a specific radioimmunoassay revealed that SN is the predominant cleavage product of secretogranin II, a member of the chromogranin/secretogranin protein family, in adult ganglia. SN was detected within the majority of nerve endings surrounding postganglionic neurons that were identified by the presence of synaptophysin and, in part, colocalized leu-encephalin. Applying immuno-electronmicroscopy, SN was localized to large dense core vesicles of neuronal and small intensely fluorescent (SIF) cells. In situ hybridization revealed the presence of secretogranin II mRNA in postganglionic neurons and, to a lesser extent, in SIF cells. One week after transection of the postganglionic branches SN levels were not significantly altered; however, a decrease of secretogranin II mRNA was observed in postganglionic neurons but not in SIF cells. After decentralization of the ganglion, SN-immunoreactive nerve terminals disappeared and intraganglionic SN levels were reduced by 70%, indicating the preganglionic origin of SN-positive nerve fibres and varicosities. Secretogranin II mRNA was slightly reduced under this condition. Combined axotomy and decentralization further diminished intraganglionic secretogranin II mRNA, although peptide levels increased significantly above control values under these conditions. Double-labelling immunofluorescence with antibodies against the somatodendritic marker microtubule-associated protein 2 (MAP2) revealed that the increase in SN immunoreactivity was due to an accumulation of SN in axonal processes of postganglionic neurons. SN immunoreactivity was also detected in dissociated neonatal superior cervical ganglion cultures and increased significantly upon treatment with nerve growth factor, the survival and differentiation factor of sympathetic neurons during perinatal development. Co-culture with non-neuronal cells or addition of leukaemia inhibitory factor, a cytokine known to stimulate synthesis of various peptides after nerve transection, did not influence SN immunoreactivity. Therefore, since no fixed relationship between SN and any of the known neuropeptides or neurotransmitters expressed in sympathetic neurons was observed, the expression of this novel peptide appears to be independently regulated.