Extrinsic adrenergic innervation of the extrahepatic biliary duct system in guinea-pigs,cats and rhesus monkeys

Summary The localization of catecholamines has been investigated in the extrahepatic biliary duct system of cats, guinea-pigs and rhesus monkeys. In fluorimetric determinations noradrenaline was found to be the main primary catecholamine present in the biliary tract of rhesus monkeys. There exist regional differences in the noradrenaline content: Fairly low amounts were detected in the lower fundus of the gall-bladder (0.28 g/g). Increasing concentrations were measured in the corpus vesicae felleae (0.35 g/g), reaching a maximum level in the collum vesicae (0.49 g/g) and the ductus cysticus (0.50 g/g). The noradrenaline content of the choledochus and the choledocho-duodenal junction including Oddi's sphincter was much lower: 0,27 and 0,25 g/g respectively. The noradrenaline level in the small intestine of the rhesus monkey amounted to less than half the concentration found in the biliary ducts. Neither dopamine nor adrenaline have been detected. Fluorescence microscopical analysis reveals the presence of adrenergic nerves in the bile ducts which correspond to the measured noradrenaline concentrations: All parts of the biliary duct system in the different species investigated contain an elaborate perivascular adventitial plexus and adrenergic fibres confined to adventitial non-adrenergic ganglia. In guinea-pigs adrenergically innervated ganglia extend into the smooth muscle layer. The smooth muscle layer of the gall-bladder and the terminal choledochus in cats and rhesus monkeys is penetrated by a wide-meshed adrenergic ground plexus. This plexus was absent in guinea-pigs. The smooth musculature of the sphincter Oddi lacks a specialized adrenergic nerve supply in all species investigated. Finally, bound to the arterial vascular bed inside the propria in all parts of the biliary tract from all species investigated a prominent perivascular plexus is present. It is concluded that the smooth musculature of the gall-bladder and the terminal choledochus (the sphincter region excluded) in cats and monkeys receives 1. a direct sympathetic noradrenergic inhibitory innervation and 2. an indirect sympathetic noradrenergic inhibitory innervation which acts on intrinsic excitatory neurons and is present in all species investigated. The functional significance of the direct and indirect inhibitory innervation to the smooth musculature of the gall-bladder is discussed in detail.Dedicated to Professor Bengt Falck.Supported by the Deutsche Forschungsgemeinschaft and Joachim-Jungius-Gesellschaft zur Förderung der Wissenschaften, Hamburg.     

Histofluorescence and histochemical analysis of adrenergic and cholinergic innervation of the lungs in burns

Neurohistochemical methods were applied to study adrenergic and cholinergic nervous structures of pulmonary tissue in 30 patients aged 14-82 who had died of burn disease at various stages of its progress (shock, toxemia, septic toxemia). Autopsies performed within 4 hours after the patients' death evidenced mediators depletion in perivascular and peribronchial plexuses as well as intensive luminescence of nervous fibers considered as compensatory adaptation. There was cholinesterase hypoactivity in cholinergic plexuses. It is established that the failure of adaptive-trophic sympathetic regulation of pulmonary tissues including relevant vessels and bronchi results in diminution of pulmonary compensatory and adaptive potential. This should be allowed for in the treatment of burn disease complications.     

Noradrenergic and adrenergic systems in the brain of the urodele amphibian,Pleurodeles waltlii,as revealed by immunohistochemical methods

The distribution of noradrenaline and adrenaline in the brain of the urodele amphibian Pleurodeles waltlii has been studied with antibodies raised against noradrenaline and the enzymes dopamine--hydroxylase and phenylethanolamine-N-methyltransferase. Noradrenaline-containing cell bodies were found in the anterior preoptic area, the hypothalamic nucleus of the periventricular organ, the locus coeruleus and in the solitary tract/area postrema complex at the level of the obex. Noradrenergic fibers are widely distributed throughout the brain innervating particularly the ventrolateral forebrain, the medial amygdala, the lateral part of the posterior tubercle, the parabrachial region and the ventrolateral rhombencephalic tegmentum. Putative adrenergic cell bodies were found immediately rostral to the obex, ventral to the solitary tract. Whereas the cell bodies and their dendrites were Golgi-like stained, axons were more difficult to trace. Nevertheless, some weakly immunoreactive fibers could be traced to the basal forebrain. A comparison of these results with data previously obtained in anurans reveals not only several general features, but also some remarkable species differences.Abbreviations Acc Nucleus accumbens - AP area postrema - Apl amygdala, pars lateralis - Apm amygdala, pars medialis - ca commissura anterior - Cb cerebellum - cc central canal - Dp dorsal pallium - epl external plexiform layer - gl glomerular layer of the olfactory bulb - H ganglion habenulae - igl internal granular layer - Ip nucleus interpeduncularis - Lc locus coeruleus - Ll lateral line lobe - Lp lateral pallium - Ls lateral septum - ml mitral cell layer - Mp medial pallium - Ms medial septum - nPT nucleus pretectalis - NPv nucleus of the periventricular organ - nV nervus trigeminus - oc optic chiasm - Poa preoptic area - Ri nucleus reticularis inferior - SC nucleus suprachiasmaticus - sol solitary tract - Str striatum - thd thalamus dorsalis - thv thalamus ventralis - To tectum opticum - TP tuberculum posterius - V ventricle - VH ventral hypothalamic nucleus - III nucleus nervi oculomotorii - IXm nucleus motorius nervi glossopharyngei - Xm nucleus motorius nervi vagi     

Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein

 We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2′-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was detectable in the ventral region of the limb primordium, and β-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium. Received: 7 April 1997 / Accepted: 15 May 1997     

Peripheral innervation pattern in myotomal muscle of two teleosts as revealed by intracellular marking

Spinal nerves of abdominal and trunk segments from cod (Gadus morhua L.) and freshwater eel (Anguilla anguilla L.) were examined by light and electron microscopy and major differences in peripheral innervation patterns investigated.
Histochemically stained frozen sections clearly resolve the focal (terminal) innervation of eel fast fibres (one endplate/fibre) and the multiple innervation pattern of cod trunk muscle (around 20 endplates/fibre). Sub junctional folds are absent from eel and poorly developed in cod neuro-muscular junctions (NMJs). A single population of synaptic vesicles with mean diameter of22–25 nm is found in the NMJs of both species.
Cobalt chloride was used as an intracellular marker to map the branching between spinal roots and fast muscle endplates. Evidence was obtained for substantial inter-segmental branching of the ventral rami in cod but not in eel. Previous classification schemes, dividing fishes according to the presence of separate or mixed motor and sensory axon populations within the ventral rami, are shown to be dependent upon sample location. In this study, the ventral rami of both species contain mixed populations, having modal diameters around 2 μm and8–12 μm, with the proportion of small diameter (sensory) axons much higher in cod than eel. The results suggest a more coarse control of body curvature in cod but an increased importance of pro-prioreceptors in generating the locomotory wave.     

Fluorescence histochemical observations on the intrinsic adrenergic innervation and monoamine-containing cells of the feline pancreas,with special reference to the terminal main pancreatic duct of wirsung

Summary Intrinsic adrenergic innervation of the pancreas of the cat was studied using two fluorescence histochemical methods. Special emphasis was focused on the monoaminergic regulation of the mechanisms responsible for dynamically active suction-pressure pumping at the choledocho-pancreatico-duodenal junction, consisting of a labyrinthine reservoir chamber surrounded by a contractile mantle of smooth muscle, described recently (Kyösola, 1976). In that junction area, a rich distribution of single varicose fluorescing nerve fibers and small nerve fascicles, both free (i.e., unrelated to the blood vessels) and forming typical perivascular nerve plexuses, as well as large ganglia of nonfluorescing nerve cells surrounded by fluorescing baskets of varicose nerves were observed in the connective, fat, and pancreatic tissues, as well as between the smooth muscle bundles of the contractile smooth muscle mantle surrounding the reservoir chamber and its minor compartments. The epithelial lining of the labyrinthine duct system contained some solitary brightly yellow fluorescing enterochromaffin cells. In addition, two other categories of fluorescing cells were observed: (1) Small rounded cells with a relatively large rounded nucleus, and exhibiting a clearly bluish, usually granular, fluorescence varying from weak to intense; (2) Larger cells exhibiting quite a weak, clearly greenish to greenish-yellow fluorescence, either gathered in homogeneous clusters or mingled with the cells of the former type into heterogeneous cells clusters. These two cell types were clearly distinguishable from each other, but intermediate cell types were also seen. Thus, a continuous scale of fluorescing cells was observed, the color of the fluorescence varying from clearly bluish to greenish-yellow, the intensity of the fluorescence varying from intense to weak (respectively, in general), and the size ranging from small to large (respectively). These cells, probably heterotopic alpha cells of the islets of Langerhans, were frequently in quite close proximity to the smooth muscle surrounding the contractile labyrinthine system or its minor compartments. On the other hand, they were frequently in close proximity to quite large thin-walled sinusoid-type blood vessels. At least some of these fluorescing cells were surrounded by fluorescing varicose nerves, suggesting the existence of a neuroendocrine link. Scattered among these fluorescing cells, and also gathered into small clusters, were nonfluorescing nerve cells interconnected with fluorescing varicose nerves. The fluorescing cells of the endocrine pancreas, similarly, could be arbitrarily classified into two main categories, although intermediate cell types were observed as well: (1) Small rounded cells with a large, ovoid to rounded, centrally placed nucleus, and exhibiting a moderate to intense granular cytoplasmic fluorescence. The color of the fluorescence varied from yellow-green to blue-green, and there were considerable differences also in the fluorescence intensity. (2) Mixed irregularly with the type (1) cells were cells with more or less the same cytologic characteristics, but slightly larger, and exhibiting a rather homogeneous weak yellow fluorescence. Sometimes the fluorescence intensity was so weak that it was hardly discernible. These cells were packed densely and irregularly with the type (1) cells within the clusters, and no clear-cut spatial organization between these two cell types was observed. In the wall of the main pancreatic duct of Wirsung, both free and perivascular fluorescing nerves were seen within the different layers, including the (distally incomplete) smooth muscle layer. The epithelial lining of the duct contained enterochromaffin cells, sometimes located so deeply in the epithelial invaginations that they were in close proximity to the smooth muscle. The distribution of fluorescing nerves in the exocrine pancreas was sparse. Typical enterochromaffin cells exhibiting a bright yellow granular cytoplasmic fluorescence were scattered either solitarily or in small groups in the epithelial lining of the acini and of the small excretory ducts. Blue-green fluorescing varicose adrenergic axons were some-times seen to connect enterochromaffin cells or groups of them located in different acini. Scattered within the parenchyme of the exocrine pancreas were clusters of nonfluorescing nerve cells surrounded by typical baskets of fluorescing varicose terminal ramifications of adrenergic axons. In the endocrine pancreas, similarly, only a relatively sparse distribution of fluorescing nerves was observed among the islet cells and surrounding clusters of them. However, groups of islet cells were observed to be interconnected by fluorescing varicose nerves coursing through or near nonfluorescing ganglia, suggesting an integrative neural connection between pancreatic islets and nonadrenergic ganglia via adrenergic nerves.     

Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids

Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of catecholamines in peripheral adrenergic nerves in stretch preparations with fluorescence induced by aqueous solution of glyoxylic acid.

Fluorescence induced by aqueous solution of glyoxylic acid and formaldehyde-induced fluorescence of catecholamines were compared for the demonstration of peripheral adrenergic nerves in stretch preparations. Glyoxylic acid was better than formaldehyde for the demonstration of the adrenergic nerves. On the other hand, the formaldehyde was better than glyoxylic acid for the demonstration of biogenic amines in cell bodies.     

Linkage arrangement in the vitellogenin gene family of Xenopus laevis as revealed by gene segregation analysis.

Using restriction fragment length polymorphism (RFLP) we have analyzed the segregation of alleles of the different vitellogenin genes of Xenopus laevis. The results demonstrate that the four genes whose expression is controlled by oestrogen, form two linkage groups. The genes A1, A2 and B1 are linked genetically whereas the fourth gene, the gene B2, segregates independently. The possible origin of this unexpected arrangement is discussed.     

Evidence for the presence of serotonin in Mysidacea (Crustacea,Peracarida) as revealed by fluorescence immunohistochemistry

In crustaceans, serotonin (5-HT) exerts a wide range of physiological actions on many tissues. However, 5-HT has not been detected to date in Mysidacea (Crustacea, Peracarida). We have investigated the presence of 5-HT in the brain and the eyestalks of two Mysida (Leptomysis lingvura, Hemimysis margalefi) and one Lophogastrida (Lophogaster typicus) species by using the immunohistofluorescence technique. 5-HT-like immunopositive areas exhibit a similar pattern in the three species. 5-HT-like immunostaining is present in the retinular photosensitive cells, except in the deep-living species L. typicus. 5-HT-like cell bodies and fibres are observed in the lamina ganglionaris and in the three medullae. In the sinus gland, only 5-HT-like endings are detected. In the eyestalks, 5-HT-like fibres detected in the optic tract link with the protocerebrum, in which 5-HT-like somata and their extensions are found. Some neurones are detected in the anterior median cell cluster, in the protocerebral bridge and in the central body. In the deutocerebrum, the paracentral lobes are connected by immunoreactive fibres that run along the deutocerebral commissure. The glomeruli of the olfactory lobes exhibit strong diffuse immunostaining. Beside and in the median part of the deutocerebrum, at least two large serotoninergic neurones project their axons into the olfactory lobe cell cluster. Immunoreactive fibres are also found in the antennular neuropiles. Our results demonstrate the presence of 5-HT-like cell bodies and fibres in Mysidacea. The distribution patterns of the 5-HT immunoreactivity found herein are compared with those of other peracarids and decapods.     

Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning electron microscopy

Summary The osmium-ligand binding technique and scanning electron microscopy have been applied to the study of the three-dimensional organization of mesocarp cells of a mature avocado fruit. Using this approach the mitochondria of the cells appear as elongated, branching structures and the endoplasmic reticulum consists of a complex of tubular strands, vesiculated strands and lamellar sheets. Associations of the endoplasmic reticulum with other organelles are also apparent. It is suggested that this approach provides a valuable means to assess the structural transitions in cell organization that occur during development or with functional changes.     

Morphology of adrenergic and cholinergic innervation of the kidney as affected by spontaneous and ischemic renal hypertension in rats

The innervation of basic renal structures (vascular glomerular pole with juxtaglomerular apparatus and renal tubules) was experimentally studied in rats at different stages of spontaneous and Goldblatt hypertension. Both fluorescent determination of adrenergic innervation and histochemical identification of cholinergic innervation have shown that they increased in spontaneous and remained unchanged in renal hypertension. The data obtained confirm an important role of intrarenal innervation in the development of kidney "resettings" in spontaneous hypertension.