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.     

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.     

铃蟾肽介导的豚鼠肠系膜下神经节非胆碱能迟慢兴奋性突触后电位

应用细胞内记录技术,对铃蟾肽(bombesin,BOM)在豚鼠离体肠系膜下神经节(inferior mesenteric ganglion,IMG)非胆碱能兴奋性突触传递中的作用进行了研究。重复电刺激突触前结肠神经,有74．3％(52／70)IMG细胞可诱发迟慢兴奋性突触后电位(ls-EPSP)。在可引出ls-EPSP的细胞中,22％(4／18)细胞同时对BOM和SP敏感。用BOM持续灌流IMG,可明显抑制对BOM敏感细胞的ls-EPSP,对BOM不敏感细胞的ls-EPSP则无影响,且BOM受体与SP受体间无交叉脱敏。BOM受体阻断剂tyr^4[D-phe^12]bombesin能明显可逆性地抑制BOM敏感细胞的ls-EPSP和去极化,但对BOM不敏感细胞则无影响。研究结果提示,BOM可能是介导豚鼠IMG细胞ls-EPSP的一种递质。     

The origin of ovarian neuropeptide Y (NPY)-immunoreactive nerve fibres from the inferior mesenteric ganglion in the pig

Summary Applying a double-immunofluorescence technique, the porcine ovary is demonstrated to receive two populations of NPY-immunoreactive nerve fibres originating from the inferior mesenteric ganglion: one with colocalized tyrosine hydroxylase and supplying predominantly the ovarian vasculature, and a second, solely NPY-immunoreactive and almost exclusively associated with growing follicles. A third group of tyrosine hydroxylase-and dopamine--hydroxylase-positive, but NPY-negative nerve fibres is associated with ovarian blood vessels and, to a minor extent, with ovarian follicles. As revealed by retrograde tracing, the vast majority of postganglionic neurons projecting to the ovary is located in a discrete area of the ganglion, suggesting a somatotopic organization of the porcine inferior mesenteric ganglion. Moreover, the finding indicate that three subpopulations of postganglionic sympathetic neurons with different chemical codes supply different target components of the porcine ovary. The physiological relevance of the described neurons in the nervous control of ovarian functions remains to be elucidated.A portion of these results has been presented in abstract form (Majewski et al. 1991)     

Projection pathways,co-existence of peptides and synaptic organization of nerve fibers in the inferior mesenteric ganglion of the guinea-pig

Summary The presence of immunoreactive enkephalin, dynorphin, vasoactive intestinal polypeptide, cholecystokinin, substance P and neuropeptide Y in nerve fibers that project to the guinea-pig inferior mesenteric ganglion was analysed, after different denervation and ligation procedures. A quantitative analysis demonstrates that enkephalin- and substance P fibers reach the ganglion mainly via lumbar splanchnic and partly via intermesenteric nerves. Dynorphin-, vasoactive intestinal polypeptide- and cholecystokinin fibers reach the ganglion mainly via colonic and partly via hypogastric or intermesenteric nerves. Neuropeptide Y fibers enter via intermesenteric, lumbar splanchnic and hypogastric nerves and pass through the ganglion. Analysis of serial 0.5 m sections tends to confirm co-existence: of dynorphin, vasoactive intestinal polypeptide and cholecystokinin in fibers projecting from the colon; of dynorphin with substance P in the lumbar splanchnic nerves; and of neuropeptide Y with substance P in the hypogastric and colonic fibers. Synaptic contacts, predominantly axodendritic, onto the ganglion cells from enkephalin-, vasoactive intestinal polypeptide-, and substance P-containing terminals were revealed by electron microscopy. Enkephalin-immunoreactive axon varicosities are filled with small, clear vesicles with a few large, cored vesicles and form asymmetric synapses; dynorphin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive axon varicosities are rich in large, dense-cored vesicles and form symmetric synapses.     

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.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

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.     

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.     

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.     

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P⁺/CGRP⁺ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P⁺/CGRP⁺ dorsal root ganglion cells. (2) Substance P⁺/CGRP^– fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP⁺/substance P^– fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are throught to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50–130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

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.     

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia. This work was supported by NIH grant HL65481 (R.L.P.) and HL54633 (D.B.H.). Use of the DeltaVision Restoration microscope was provided through the Imaging/Physiology Core supported by NIH Grant P20 RR16435 from the COBRE program of the National Center for Research Resources     

Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat

Summary The cellular localization of carbonic anhydrase (CAH) in the carotid body of the rat was investigated by means of Hansson's cobalt-precipitation technique in cultures of dissociated cells. In both young (2-day-old) and old (77-day-old) cultures, the parenchymal glomus (type-I) cells were selectively stained by this technique, and in addition expressed tyrosine hydroxylase and neuron-specific enolase as revealed by immunofluorescence. Enzymic reaction product of CAH appeared to be predominantly intracellular since staining was more intense and occurred more rapidly following permeabilization of the cell membranes with Triton X-100; its formation was inhibited by the CAH-inhibitor acetazolamide (1–10 M) or by increasing the pH from 5.8 to 7.5. Cryostat sections of the carotid bifurcation revealed intense CAH-reaction product in cell clusters of the carotid body, in a few cells of the nodose ganglion, and in red blood cells. Neuronal cell bodies of the petrosal ganglion and superior cervical ganglion (SCG) were largely non-reactive. The SCG is known to contain clusters of small intensely fluorescent (SIF) cells, which were also non-reactive when grown in dissociated cell culture. Thus, although glomus and SIF cells are often considered to be similar cell types, functional CAH-activity appears unique to glomus cells, and this may be important for the physiological response of the carotid body to certain chemosensory stimuli.     

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998     

Immunological identification of soluble carbonyl reductase with testosterone 17β-dehydrogenase (NADP+) in guinea pig liver and kidney

Antiserum was produced against one of two carbonyl reductases purified from guinea pig liver cytosol to identify the enzymes as testosterone 17β-dehydrogenase isozymes. Immunoelectrophoresis and immunoprecipitation with the antiserum indicated that the two reductases had common antigenic sites. The antiserum inhibited most of both carbonyl reductase and testosterone 17β-dehydrogenase activities in the purified reductases and in cytosols of liver and kidney.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion

The origin of nitric oxide synthase-containing nerve fibers in rat celiac-superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the cceliac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons.