Adrenergic retinal neurons

Summary Adrenergic retinal neurons have been studied in cynomolgus monkeys, cats, rabbits, guinea-pigs, rats, and mice with the fluorescence technique of Falck and Hillarp. With some species variations, three adrenergic fibre layers have been observed: an outer adrenergic fibre layer (all species) at the border between the inner nuclear and inner plexiform layers, a middle adrenergic fibre layer (rabbits, guinea-pigs, rats, and mice) in the middle of the inner plexiform layer, and an inner adrenergic fibre layer (rabbits) at the border between the inner plexiform layer and the ganglion cell layer. Similarly, three kinds of adrenergic nerve cells have been found: a somewhat heterogenous group of outer adrenergic cells (all species) situated in the innermost cell rows of the inner nuclear layer, eremite cells (rabbits, guinea-pigs, rats, and mice) within the inner plexiform layer and alloganglionic cells (all species) with a position and appearance resembling some of the ordinary non-adrenergic cells of the ganglion cell layer. All the adrenergic cells are star-shaped with slender branching processes running to the different adrenergic layers.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 66-14 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant no. NB 05236-02), by the Swedish Medical Research Council (grant no. B 66-320), and by the Faculty of Medicine, University of Lund, Sweden.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.)

Summary The localization and intraneuronal distribution of the monoaminergic transmitters in the nervous system of the earthworm, Lumbricus terrestris, have been investigated in detail with the aid of the histochemical fluorescence method of Falck and Hillarp.In the ventral nerve cord, many yellow fluorescent, 5-hydroxytryptamine containing neurons are found, but only few green fluorescent noradrenaline containing cell bodies, which, however, are numerous in the peripheral nervous system. There is an abundance of both fibre types in the neuropile.The 5-hydroxytryptaminergic neurons probably have a motor (possibly inhibitor) function; the adrenergic neurons in the body segments are supposed to have a receptor (exteroceptive and possibly proprioceptive) function.In the cerebral ganglion, both 5-hydroxytryptamine and noradrenaline containing neurons are found in large numbers, and there are closely packed numerous fibres of both types in the neuropile. Their function is more obscure, though an associative function can be presumed for some adrenergic neurons; smaller 5-hydroxytryptaminergic neurons might have a motor (perhaps inhibitor) function.Adrenergic sensory cells are found in the body integument, most frequently in the clitellum segments, in the prostomium, and in the roof of the buccal cavity. These cells give off varicose fibres that form a basi-epithelial network which is in communication with the green fluorescent sensory fascicles in the ventral nerve cord via the epidermal nerves, the ring nerves, and the segmental nerves. No direct adrenergic sensory-effector innervation of either circular and/or longitudinal musculature or gland cells seems to exist. No adrenergic free nerve endings in the body integument have been observed. Instead, there must be a synaptic contact with the motoneurons, either directly in the neuropile or via an interjacent neuron.No synaptic contacts have been observed in the ventral nerve cord between adrenergic or 5-hydroxytryptaminergic fibres and either the giant fibres or fluorescent or nonfluorescent perikarya.An adrenergic innervation of the pharynx musculature has been found, and sensory cells of a different type are present in and below the epithelium; here, a direct senso-motoric innervation of the pharyngeal musculature cannot be excluded. It is established that the adrenergic neurons in the stomatogastric nervous system have an exciting function on the pharynx, whereas a direct monoaminergic influence of the muscular movements of the intestine probably does not exist.Abbreviations Used A adrenaline - CA catecholamine - DA dopamine - 5-HT 5-hydroxytryptamine - MA monoamine - NA noradrenaline The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the Swedish Natural Science Research Council (99-34, 6627), and by the Swedish Medical Research Council (B67-12X-712-02A).     

Adrenergic and cholinesterase-containing neurons of the heart

Summary The adrenergic and acetylcholinesterase-containing nerves of the hearts of mice, rats, guinea-pigs, rabbits, and cats were studied. The fluorescence technique of Falck and Hillarp was used for the demonstration of adrenergic nerves, whereas a modified Koelle cholinesterase technique was used for the cholinesterase-containing nerves. The inhibitors used were Mipafox, iso-OMPA and Nu 683. Microspectrofluorometry was used to identify the structures containing dopamine.Adrenergic as well as acetylcholinesterase-containing fibres were found in all parts of the heart, most abundantly in the atria. Dense nerve plexa supplied the sinoarial and atrioventricular nodes. There was a plexus of both fibre types in the endocardium and on the atrial side of the valves. In the valves, it could be shown that adrenergic and cholinesterase-containing fibres ran closely parallel to each other. Indirect evidence suggested that this applies also to the myocardium.No nerve fibres containing dopamine were revealed in the microspectrofluorometer. The dopamine previously found in the atria seems, instead, to be situated in so-called small intensely fluorescent cells.No adrenergic ganglion cells were found in the heart despite extensive search. The vagus of rabbits was found to contain only few adrenergic preterminals.     

Neuronal localization of dopamine and 5-Hydroxytryptamine in some mollusca

Summary The localization of biogenic monoamines in ganglionic tissues from Anodonta piscinalis, Helix pomatia, and Buccinum undatum has been studied by means of the histochemical fluorescence method of Falck and Hillarp.In cerebral, visceral, and pedal ganglia (besides nonfluorescent nerve cells) neurons emitting a green or yellow fluorescence were found. No other cell systems exhibiting a specific fluorescence were observed. An abundance of monoaminergic terminals were found in the central parts of these ganglia. Spectrophotofluorimetric determinations showed that there are large quantities of dopamine and 5-hydroxytryptamine in the tissues investigated. The amounts of dopamine and 5-hydroxytryptamine agree well with the distribution of green and yellow fluorescence, respectively, in the ganglia.There are many similarities between the vertebrate and the molluscan monoaminergic neurons. The morphology of the neurons is the same, the intraneuronal distribution of the monoamines is identical, depletion experiments with reserpine and denervation experiments give the same results, and the synaptic arrangement of monoaminergic fibres on non-adrenergic neurons has the same appearance. Apparently, however, dopamine and 5-hydroxytryptamine are the only monoamines acting as neuronal transmitters in the species investigated.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 64-5 through the European Office of Aerospace Research (OAR), United States Air Force and by the Swedish Natural Science Research Council.     

Adrenergic innervation of the oesophagus in the cat (Fellis domestica) and rhesus monkey (Macacus rhesus)

Summary The distribution of monoamines in the pharynx and oesophagus of the rhesus monkey (Macacus rhesus) and the cat (Felis domestica) was investigated by means of fluorescence microscopical and chemical methods. Fluorimetric determinations reveal the presence of varying amounts of noradrenaline in the pharynx and oesophagus of the rhesus monkey. The lowest amount (0.05 (g/g) was found in the lower part of the oesophagus, the so-called sphincter-segment. The middle and upper part of the oesophagus contain medium amounts of noradrenaline (0.06–0.09 g) whereas the highest concentration was detected in the pharynx (0.14 (g/g). Neither dopamine nor adrenaline occurred in the tissue pieces analyzed. Fluorescence microscopically noradrenaline was found to be located in varicose intramural nerve fibre plexus which innervate mucous glands and blood vessels in the pharynx of both species. In the rhesus monkey, the lamina muscularis mucosae of all parts of the oesophagus is supplied by a well developed noradrenergic ground-plexus. Preterminal and terminal varicose nerve fibres are distributed in myenteric and submucous ganglia of the oesophagus; the number of such ganglia decreases towards the lower segment. The density of the adrenergic innervation is higher in myenteric when compared to submucous ganglia. The arrangement of the intraganglionic terminals suggests that both axosomatic and axodendritic contacts occur in Auerbach's ganglia whereas axodendritic contacts seem to predominate in Meissner's ganglia. Myenteric ganglia situated close to the submucosa as well as true submucous ganglia may be occasionally seen to be traversed by faintly fluorescent non-varicosed fibres which do not establish any synaptic contacts. The fluorescence intensity of intraganglionic varicosities varies considerably; accordingly the transmitter content of individual varicosities seems to be very variable. The adrenergic innervation of the lamina muscularis is restricted to single contorted fibres being sparsely distributed throughout the longitudinal smooth muscle layer. The circularly arranged smooth musculature of the sphincter-segment lacks an adrenergic nerve supply. The vagus nerve carries sympathetic adrenergic fibres to the lower oesophagus and the cardia. Species differences between the innervation pattern in rhesus monkeys and cats are outlined: No adrenergically innervated ganglia occur in the submucosa of the cat. However, part of the myenteric ganglia in cats exhibit an adrenergic innervation pattern similar to that seen in submucous ganglia of the rhesus monkey. They might therefore be regarded as morphologically equivalent to the plexus submucosus which is, however, present in the whole gut. The density of the noradrenergic ground-plexus in the muscularis mucosae of the cat's oesophagus is less than that of the corresponding plexus in rhesus monkeys.The influence of noradrenaline upon the smooth musculature and the neurons from myenteric as well as submucous ganglia is discussed. From the point of view of the adrenergic innervation there is no structure corresponding to the sphincterlike lower oesophageal segment.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius-Gesellschaft zur Förderung der Wissenschaften, Hamburg.     

Localization of monoaminergic neurons in the central nervous system of Astacus astacus Linné (Crustacea)

Summary The cellular localization of biogenic monoamines in crustaceans was studied by means of a highly specific and sensitive fluorescence method devised by Falck and Hillarp. It was found that neurons displaying specific fluorescence in the central nervous system were confined to the protocerebrum, the medulla externa and interna and the ventral nerve cord. The method allows a distinction between the fluorophores of 5-hydroxytryptamine (and 5-hydroxytryptophan), which emit the yellow light, and the fluorophores deriving from the catecholamines (and DOPA), which emit the green light. Green-fluorescent neurons occurred abundantly in the aforementioned parts of the central nervous system while yellow-fluorescent neurons were sparsely present in the same parts.The present work has been carried out at the departments of Histology and Zoology at the University of Lund. The authors take great pleasure in expressing their warmest thanks for laboratory facilities, provided by Professors Erik Dahl (Zoological Institute) and Bengt Falck (Histological Institute).The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 66-14 through the European Office of Aerospace Research (OAR), United States Air Force and by a grant from the Swedish Natural Science Research Council 99-32 (nr 5995).     

A method for the demonstration of adrenergic nerve fibres in peripheral nerves

Summary The adrenergic nerve fibres running from the ganglia to the innervated tissues usually have too low a content of noradrenaline to be clearly visualized with the histochemical fluorescence method of Falck and Hillarp. They can easily be demonstrated, however, as early as 24 hours after axotomy (crushing or constriction of the nerves) due to the rapid accumulation of what is probably noradrenaline taking place proximally to the lesion. The fibres can be visualized even more clearly if axotomy is combined with the administration of l-dopa and with monoamine oxidase inhibition. In this way the presence, distribution and direction of adrenergic fibres can be directly studied in peripheral nerves.For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine) and Swedish Pfizer, Stockholm (nialamide). The investigation has been supported by research grants from the United States Public Health Service (NB 02854-04), the Swedish Medical Research Council, and Knut and Alice Wallenbergs Foundation.     

Cholinergic innervation of the pancreas in the sheep

Antibodies raised against vesicular acetylcholine transporter (VAChT) were applied to study the cholinergic innervation pattern of the pancreas of the sheep. To determine whether the cholinergic pancreatic neuronal elements contain tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) or substance P (SP) double immunocytochemistry was used. A moderate number of VAChT-immunoreactive (IR) nerve terminals were distributed between the acini, whereas only single cholinergic nerve fibres innervated the interlobular connective tissue. VAChT-positive nerve fibres supplying the endocrine pancreas were found only occasionally. The pancreatic blood vessels and ducts system were devoid of VAChT-containing nerve endings. All intrapancreatic neurons studied showed immunoreactivity to VAChT, but intrapancreatic ganglia were not innervated with cholinergic nerve fibres. The colocalization of VAChT and TH or VAChT and SP was detected in distinct populations of nerve fibres localized amongst the acini, but not within the islet nor in the connective tissue. Single VAChT-IR nerve terminals co-expressing NPY were distributed around the acini, islets as well as in the connective tissue septa. A moderate number of VAChT-IR/VIP-IR nerve endings were located in the exocrine pancreas, whereas the islets and connective tissue were innervated with VAChT/VIP-containing nerve fibres only occasionally. In the vast majority of VAChT-positive intrapancreatic perikarya the presence of TH was additionally found. A moderate number of VAChT-IR intrapancreatic perikarya co-expressed NPY, SP or VIP. The results of the present study demonstrate species-dependent cholinergic innervation pattern of the pancreas of the sheep. The co-localization of VAChT with the neuropeptides suggests the existence of functional interactions influencing the ovine pancreas (mainly exocrine) activity.     

Degeneration of adrenergic nerves in the urinary bladder during pregnancy

The adrenergic innervation of the urinary bladder of normal female and pregnant rats has been studied using a fluorescence histochemical method. The bladder is richly innervated by adrenergic nerve fibres as is evidenced by the presence of numerous adrenergic nerves in the adventitia, musculosa and submucosa. However, adrenergic nerve cells could not be observed. During pregnancy, adrenergic nerve fibres showed signs of degeneration, as most of the nerve fibres disappeared and the surviving fibres were much swollen. 10 days after parturition the pattern and density of adrenergic innervation became almost similar to those of the control animals.     

Adrenergic innervation of the salivary glands in the rat

Summary The adrenergic innervation of the major salivary glands in the rat has been studied by a specific histochemical method for the visualization of the adrenergic transmitter. Adrenergic varicose nerve fibres were found, located in a typical adrenergic ground plexus closely surrounding the serous acini of the submaxillary and parotid glands, but not the acini of the mainly mucous sublingual gland. The ducts were found to be completely devoid of adrenergic innervation. Arterioles and venules in the stroma of all three glands and certain very small vessels, possibly the sphincters of arterio-venous anastomoses, were also richly innervated by adrenergic vasomotor fibres. The relationship of the adrenergic nerve fibres to the different functional units of the gland parenchyma is discussed.The investigation has been supported by a research grant (B 66–257) from the Swedish Medical Research Council and by a Public Health Service Research Grant (NB 05236-01) from the National Institute of Neurological Diseases and Blindness.     

Pituitary adenylate cyclase-activating polypeptide in intrinsic and extrinsic nerves of the rat pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.     

Effects of light and dark upon photoreceptor synapses in the retina of Xenopus laevis

Summary The adrenergic innervation of the juxtaglomerular complex was studied in kidneys from mice, rats, guinea-pigs, rabbits, cats, dogs, pigs, monkeys, and humans using fluorescence histochemistry of neuronal nor-adrenaline and autoradiography of ³H-noradrenaline. The localization of the nerves was established by phase contrast optics or by perfusing the vascular system with India ink. Adrenergic nerve terminals, exhibiting a formaldehyde-induced fluorescence and having the ability to take up and accumulate ³H-noradrenaline, were easily identified when they enclosed the glomerular afferent arteriole. They continued in between and close to the macula densa and lacis cells to supply the glomerular efferent arteriole. The nerves could be seen to accompany this arteriole for a considerable distance until they branched off to the vasa recta in the juxtamedullary region and to adjacent cortical veins. This innervation pattern was found to be a constant feature except in kidneys from guinea-pigs and cats, in which post-glomerular adrenergic nerves were not found in some of the superficial glomerular units. The fluorescence in all adrenergic fibres supplying the juxtaglomerular complex disappeared after removal of the aortico-renal ganglion, showing that they belong to a common system of renal sympathetic nerves.This work is dedicated to Professor Wolfgang Bargmann in honour of his seventieth birthday, January 26, 1976     

Galanin-immunoreactive nerves in the mouse and rat pancreas

Summary Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin-immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non-adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas.     

Cholin- and adrenergic components of the vegetative innervation of the dura mater]

Cholin- and adrenergic nerves in the fornix and base of the dura mater of rats, cats and dogs have been studied by methods of Kelle, Falck and Glenner. It has been established that the dura mater has a developed cholin- and adrenergic nervous apparatus innervating arteries, veins and the connective tissue of the mater. The concentration of nerve fibres is always greater on a meningea media and its daughter branches. The statistical processing of the data obtained has shown that the maximum quantity of nerve fibres is in cats, less in dogs and still less in rats. It has been established that in the dura fornix of cats and dogs there are more cholinergic nerve fibres than in the base. In rats there is no such difference. The amount of fibres with monoaminoxidase approximately corresponds to the amount of conductors with noradrenaline.     

Adrenergic Nerves in the Avian Eye and Ciliary Ganglion

Summary The distribution of adrenergic fibres to the eye and to the ciliary ganglion was studied in pigeons, chicken and ducks with the aid of the sensitive and highly specific fluorescence method of Falck and Hillarp. In some animals the intensity of the fluorescence was increased by treating the animals with Nialamide and 1-DOPA. The cornea contained no adrenergic fibres except at the limbus, where a plexus of adrenergic varicose fibres was seen, partly associated with vessels. In the chamber angle, adrenergic varicose fibres were common in the loose connective tissue covering the canal of Schlemm. The canal of Schlemm was supplied by only few adrenergic fibres, but such fibres appeared along the intrascleral aqueous drainage vessels. In the iris, adrenergic varicose fibres appeared immediately in front of the posterior layer of pigment cells, strongly indicating the presence of a dilator homologous with that seen in mammals. The frontal third of the stroma contained several adrenergic varicose fibres, many of which seemed to lack association with any vessel. Varicose adrenergic fibres were also sparsely seen in the striated muscle of the iris. The ciliary processes contained many adrenergic varicose fibres, at least part of which seemed to be associated with the ciliary epithelium. The striated muscles of the ciliary body contained adrenergic varicose fibres along the vessels only. The retina contained adrenergic varicose fibres in three layers in the inner plexiform layer. Adrenergic ganglion cells of two sizes were detected in the inner nuclear layer. The retinal vessels had no adrenergic nerve fibres. The pecten was also devoid of adrenergic nerve fibres, except along the vessels close to the papilla. The optic nerve contained adrenergic varicose nerve fibres along vessels only. In the ciliary ganglion, varicose adrenergic fibres appeared at the small ganglion cells, often forming baskets of synaptic character.Acknowledgements. The work has been supported by the United States Public Health Service (grant NB 06701-01), by the Swedish Medical Research Council (project B 67-12 X-712-02 A) and by the Faculty of Medicine, University of Lund, Sweden.     

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release. Work in this study was funded by the National Health and Medical Research Council (grant numbers: 114215 and 216704; Senior Research Fellowship to B.S.), a Melbourne University Research Scholarship and the Murdoch Children’s Research Institute.     

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.     

Immunohistochemical study on the distribution of vasoactive intestinal polypeptide (VIP) containing nerve fibers in the chicken pancreas

The distribution of vasoactive intestinal polypeptide (VIP) containing nervous elements in the chicken pancreas was immunohistochemically investigated by light microscopy. Strongly VIP immunoreactive ganglia existed in the interlobular connective tissue. Ganglion containing both VIP immunoreactive and non-immunoreactive nerve cells was occasionally observed in the connective tissue. Almost all the ganglion cells also showed acetylcholinesterase (AChE) activity. No extrapancreatic nerve bundles containing VIP immunoreactive nerve fibres were detected. VIP immunoreactive nerve fibres formed plexuses in the subepithelial layer of secretory ducts and the muscle layer of small arteries. The distribution pattern of VIP immunoreactive nerve fibers was similar to that of AChE-positive nerve fibers on adjacent sections. The exocrine pancreas received a rich supply of varicose nerve fibers showing VIP immunoreactivity. B-islets also were richly innervated by VIP immunoreactive varicose nerve fibers, whereas A-islets, only poorly. These observations suggest that VIP containing nerves in the chicken pancreas have an intrinsic origin, are probably derived from VIP immunoreactive, intrapancreatic ganglion cells and innervate secretory ducts, arteries, acinar cells and B-islets, and that VIP must coexist with acetylcholine in the nervous elements.