Innervation of iridic melanophores

Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.This work has been supported by grants from Lunds Läkarsällskap, the Swedish Medical Research Council (Project no. B69-14X-2321-02 and B69-14X-712-04C) and NIH (06701-02).     

Adrenergic retinal neurons of some new world monkeys

Summary The retina of Aotes monkeys, Cebus monkeys, squirrel monkeys, and marmosets were investigated. Adrenergic perikarya were found in the innermost cell rows of the inner nuclear layer of all the investigated species. In addition, the Cebus monkey was found to have a special type of adrenergic neurons in the inner nuclear layer. This cell type was called the adrenergic pleomorph cell. Its processes ramify in the inner nuclear and inner plexiform layers. Adrenergic terminals occur in three more or less well developed sublayers of the inner plexiform layer, the layers being best developed in the Cebus monkey. Adrenergic terminals were also found around the cells of the inner nuclear layer and at the horizontal cells, where a scant sublayer is formed. More than one adrenergic sublayer of the inner plexiform layer has not been observed in primates previously, nor have the adrenergic terminals in the inner nuclear layer been observed previously in any species. The adrenergic pleomorph cells of the Cebus monkey also seem to be unique. The marked differences even between animals as closely related as some platyrhine monkeys call for caution when comparing the detailed function of the retina in different animals.This study was supported by grants from the Swedish Medical Research Council (B69-14X-2321-02) and the Faculty of Medicine, University of Lund, and was carried out within a research group sponsored by the Swedish Medical Research Council (projects No. B69-14X-56-05C and B69-14X-712-04C).     

Fluorescence microscopical demonstration of 5-hydroxydopamine in adrenergic nerves

Summary It is demonstrated that 5-hydroxydopamine (5HDA) is capable of forming highly fluorescent products with formaldehyde under conditions suitable for histochemical analyses. Prom absorptiometric and fluorometric studies it is reasonable to assume that the fluorophores formed are isoquinolines, in analogy with the reaction between, e.g., dopamine and formaldehyde. The reaction proceeds readily in protein layers and actual nerve terminals in tissues, allowing demonstration and identification of 5HDA in tissue structures. From studies on rat irides it is demonstrated that only adrenergic terminals take up 5HDA in demonstrable amounts.This work was supported by grants from the Swedish Medical Research Council (B69-14X-712-04C and B69-14X-2321-02), Knut och Alice Wallenbergs stiftelse and the Faculty of Medicine, University of Lund.     

Adrenergic and cholinergic nerves of bovine,guinea pig and hamster salivary glands

Summary The distribution of formaldehyde-induced fluorescence and acetylcholine-esterase (AChE) activity was histochemically investigated in certain salivary glands of the cow (submandibular gland), guinea pig and hamster (submandibular and sublingual glands). Adrenergic nerves occur around the secretory acini of the bovine, guinea pig and hamster submandibular glands, as well as around those of the hamster sublingual gland. The mucous secretory acini of the guinea pig sublingual gland, however, seem to be devoid of adrenergic nerve supply. Except in the sublingual gland of the hamster, no adrenergic nerves occur in relation to duct cells.The pattern of AChE activity is similar to that of adrenergic nerves. Thus, AChE-positive nerves form a network around secretory acini of all the five glands examined. Furthermore, AChE activity was also observed in nerve fibres in close proximity to striated duct cells.Both adrenergic and AChE-containing fibres were observed around blood vessels of different sizes. Ganglionic cells are occasionally to be seen; they all display AChE-activity. No adrenergic ganglionic cells were observed in any of the glands examined.All glands were also studied in the electron microscope. Interest was focussed on the fine structure of the autonomic nerves with special reference to their contents and type of storage vesicles.The content of noradrenaline was chemically determined in each type of salivary gland studied.This work was supported by grants from the University of Umeå and from the Swedish Society for Medical Research and was also carried out within a research organization supported by the Swedish Medical Research Council (projects B73-04X-712-08C and B73-04X-56-09C). The authors are indebted to Miss Kristina Karlsson and Miss Marianne Borg for valuable technical assistance.     

The arrangement of neurosecretory and catecholamine fibres in relation to the pituitary intermedia cells of the skate,Raja radiata

Summary The innervation pattern of the intermediate lobe of the skate (Raja radiata) was studied with histological and fluorescence histochemical methods. Neurosecretory fibres, stained with i.a. pseudo-iso-cyanine, were found running in bundles in the central parts of the cell cords. They terminated partly around the perinuclear parts of the intermedia cells, partly around the apices of the cells close to the vascular walls.A catecholamine innervation of the intermedia was also established. Catecholaminecontaining fibres with the appearance of nerve terminals were found around the intermedia cell apices close to the vessels. In some specimens, catecholamine fibres also seemed to terminate at the perinuclear parts of the cells.Thus it is possible, judging solely from structural relations, that both the cell body (the synthesis pole) and the cell apex (the release pole) receive a dual innervation. Recent experimental evidence indicates that the release of MSH from the pars intermedia is controlled by catecholamine fibres, but as yet there is only structural evidence for a special control of hormone synthesis.This study was supported by grants from the Swedish Natural Science Research Council (No. 99-35 and 2126-2) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-712-05 and B70-14X-56-06).     

Electron dense plaques (calcium binding sites) in the plasma membrane of the endocrine cells in rodent pancreatic islets and parathyroid glands

Summary Small electron dense plaques (EDP) were found in the plasma membrane of pancreatic islet A-, B- and D-cells and parathyroid chief cells of mice and gerbils. The identification of the EDP was facilitated by the use of special fixation techniques. The EDP may represent sites of calcium binding in the cell membranes.Supported by grants from the Swedish Medical Research Council (Project No. B76-12X-00718-11B)     

A Catecholamine storage in the pancreatic A2-cells of owl monkey (Aotes trivirgatus)

Summary The fluorescence method ofFalck andHillarp was used to study the occurrence of biogenic monoamines in the islets of Langerhans of monkeys. A storage of a catecholamine was demonstrated in the A₂-cells of owl monkey, whereas no histochemically demonstrable amount of monoamines could be seen in the islet cells of marmoset,Rhesus monkey, squirrel monkey, andCebus monkey.Supported by grants from the Swedish Medical Research Council (No. B69-14x-712-04C) and by the National Institutes of Health (No. 06701-02).     

Development of the ocular adrenergic nerve supply in man and guinea-pig

Summary The development of adrenergic nerves to the anterior eye segment was studied in human and guinea-pig embryos. Adrenergic terminals had already appeared in the earliest human embryos available (4–6 cm). They first appeared mainly in nerve trunks in the primitive chorioid, especially in the region of the developing ciliary body. Adrenergic nerves then grow into different structures of the eye as these develop, but typical terminals in contact with effector cells appeared late during the development, about the 25–30 cm stage. No adrenergic nerves were observed in the chamber angle. Corneal adrenergic nerves (also intraepithelial terminals) appeared much more frequently in embryos than in adults. No adrenergic neurons were observed in the retina. In the guinea-pig, the first adrenergic fibres were observed at about gestation day 35. The general principle of the development was very similar to that of the humans. At gestation day 45 to 50, the supply of adrenergic fibres was essentially that of the adult animal, except that the corneal adrenergic fibres were increasing until just before birth and that the adrenergic terminals of the chamber angle appeared shortly before term.This work was supported by grants from the Association for the Aid of Crippled Children, H. Hiertas Stiftelse, and the Swedish Medical Research Council (Project no. B71-14X-2321-05B).     

Neuronal localization of dopamine,noradrenaline, and 5-hydroxytryptamine in the central and peripheral nervous system ofLumbricus terrestris (L.)

Summary Fluorescence microscopical studies with the procedure of Falck and Hillarp have confirmed previous observations concerning the appearance of neurones with green and yellow specific fluorescence in the central and peripheral nervous system ofLumbricus terrestris.Chemical estimates show that the fluorescent neurones contain the primary catecholamines dopamine and noradrenaline, in addition to an indolamine, presumably 5-hydroxytryptamine. Rude's opinion that dopamine is present in a concentration twice that of noradrenaline is confirmed.Microspectrofluorometric analyses of the neurones displaying green specific fluorescence show two types of neurones, one presumably containing dopamine (mainly the receptor cells, certain small and some of the large cells in the cerebral ganglion). Some of the large cells of the cerebral ganglion and the bipolar cells near the base of the second segmental nerve in the ventral nerve cord show characteristics compatible with the simultaneous presence of both noradrenaline and dopamine in them.This work was supported by grants from the Helge Ax:son Johnson Foundation and was carried out within a reasearch organization sponsored by the Swedish Medical Research Council (projects No. B71-14X-2321-04A, B71-14X-712-06A, and B71-14X-56-07A).     

Adrenergic neurons in the spinal cord of the pike (Esox lucius) and their relation to the caudal neurosecretory system

Summary The lower spinal cord including the caudal neurosecretory system of the pike (Esox lucius) was investigated by means of light and electron microscopy and also with the fluorescence histochemical method of Falck and Hillarp for the visualization of monoamines. A system of perikarya displaying a specific green fluorescence of remarkably high intensity is disclosed in the basal part of the ventrolateral and lateral ependymal lining of the central canal. The area corresponding to the upper half of the urophysis has most cells; their number decreases caudally and cranially. A considerable number of their beaded neurites reach the neurosecretory neurons by different routes but are only occasionally present in the actual neurohemal region. An intensely fluorescent dendritic process is sometimes observed terminating with a bulbous enlargement at the ependymal surface in the central canal. Besides small, electron lucid vesicles in the terminal parts of the axons, the neurons contain numerous large dense-core vesicles which can apparently take up and store 5-hydroxydopa (5-OH-dopa) and 5-hydroxydopamine (5-OH-DA). These neurons are thought to be adrenergic and to contain a primary catecholamine, possibly noradrenaline.The varicosities of the adrenergic terminals are repeatedly observed contiguous to some of the neurosecretory axons, the membrane distance at places of contacts generally ranging from 150–200 Å. Another type of nerve terminals that contain only small empty vesicles, also after pretreatment with 5-OH-dopa or 5-OH-DA, are frequent among the neurosecretory neurons. These axons establish synaptic contacts with membrane thickenings on most of the neurosecretory neurons. Thus it seems that the neurosecretory neurons are innervated by neurons morphologically similar to cholinergic neurons and that part of them receive an adrenergic innervation, which supports the view hat the caudal neurosecretory cells do not constitute a functionally homogeneous population.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius Gesellschaft zur Förderung der Wissenschaften, Hamburg.Supported by the Swedish Natural Research Council (No. 99-35). This work was in part carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-56-06 and B70-14X-712-05).Supported by the Deutsche Forschungsgemeinschaft and USPHS Research Grant TW 00295-02.     

Adrenergic neurons in teleost retina

Summary The adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.The research reported in this document has been sponsored by USPHS Grant No. 06092 and by a Research Professorship from Research to Prevent Blindness, Inc. to A.M.L. and by the Swedish Medical Research Council (B69-14X-712-04C and B68-14X-2321-01).     

Biogenic monoamines in Hirudo medicinalis

Summary The distribution of certain catecholamines and indoleamines in the ventral nerve cord and the body segments of the medical leech, Hirudo medicinalis, was studied with the fluorescence microscope technique of Falck and Hillarp, with microspectrofluorometry, and with chemical determinations of the amines. The six cells of the segmental ganglia previously shown to be chromaffin were found to contain an amine, most probably 5-hydroxytryptamine. In the two giant cells, the amine was found on the surface of coarse intracellular granules, lying mainly at the cell membrane, and at the nucleus. The two giant cells send their axons to the body muscles, which thus seem to have a 5-hydroxytryptaminergic innervation. The four smaller amine-containing cells of the segmental ganglia send their axons to the neuropil of the ganglion.The only cell type found to contain a catecholamine (probably noradrenaline) was situated in the anterior segmental nerve in the cell cluster anterior of the nephridial duct, one cell in each nerve. The axon of this cell terminates in two or more segmental ganglia; thus these neurons seem to be afferent.This work was supported by grants from the Swedish Natural Science Research Council (project no. 99-35) and the Swedish Medical Research Council (projects no. B 68-12 X-712-03 B and B 68-14 X-56-04 B).     

The ultrastructure of the liver in thyroid-fed rats

Summary Rats were fed on a 25% casein diet or the same diet supplemented with desiccated thyroid. The rats were killed after 16 days. Histological sections of the livers of the control rats show coarse, basophilic inclusions and abundance of glycogen in the cytoplasm. In the thyroid-fed rats there is a diffuse, cytoplasmic basophilia with basophilic rods and no or almost no glycogen. Under the electron microscope large areas of glycogen are to be seen in the cytoplasm of the control animals. Mitochondria and rough-surfaced endoplasmic membranes, often in large stacks, are found together. The liver cells of the thyroid-fed rats have little or no glycogen in their cytoplasm. Mitochondria, endoplasmic reticulum, and free ribosomes and polysomes are evenly distributed all over the cytoplasm. There seems to be an increase in the ratio of free to membrane-bound ribosomes and polysomes in the thyroid-fed rats. The possible significance of this observation in relation to RNA synthesis is discussed.This work was supported by grants from the Swedish Medical Research Council (Project No. K-69-12X-623-05) and the Swedish Cancer Society (Project No. 95-K69-03X).     

Monoamine-containing neurons in the optic ganglia of crustaceans and insects

Summary With the fluorescence method of Falck and Hillarp, the presence and localization of monoaminergic neurons in the optic ganglia of several crustaceans and insects have been investigated. It was found that in both classes the monoaminergic terminals, when present, appeared (especially in the medullae externa and interna of the crustaceans and the medulla of the insects) in strata specific for each species. So far, the only monoamine (visualized by this technique) present in the crustacean optic ganglia is dopamine, whereas in the Insecta, the catecholamines dopamine and noradrenaline, and the indolamine, 5-hydroxytryptamine, are found in the optic lobe. But in the Insecta, different species show different content of these amines.This work was supported by grants 2760-3 and 2760-4 from the Swedish Natural Science Research Council (R.E.), by a fellowship from Deutsche Forschungsgemeinschaft, and a grant from the Swedish Medical Research Council B72-14X-712-D7B (N.K.). We are very grateful to the director of the Department of Histology, Faculty of Medicine, Lund, Professor Bengt Falck, who put all his facilities and knowledge at our disposal.     

Tubular cytoplasmic bodies in pancreatic islet B-cells of mice

Summary Tubular bodies of varying length and thickness are found in the cytoplasm of pancreatic islet B-cells of obese-hyperglycemic mice and a few of their lean litter mates. Most of these bodies are elongated with tapered ends. There are also some rounded or peculiarly formed variants. They are composed of numerous small electron dense tubular units, often in parallel arrangement. The tubules are embedded in a moderately dense matrix and their interior shows also moderate density. Smaller or larger electron opaque rounded particles are seen in some of the cytoplasmic bodies. Tubular bodies sometimes occur in association with mitochondria, indicating that they might be derived from these cellular organelles. Though the chemical composition and significance of the tubular bodies still are unknown, mitochondrial changes, possibly related to altered metabolic activity, are suggested to form the basis of their development.This work was supported by grants from the Swedish Medical Research Council (Project No. B69-12X-718-04A).     

Spectral absorption by screening pigment granules in the compound eye of butterflies (Heliconius)

Summary The spectral absorption by single granules, clusters and masses of granules of the screening pigment in the compound eye of the butterfly genusHeliconius was studied by microspectrophotometry. Most of the pigment granules were found to have an almost constant absorption in the wavelength region 300 to 700 nm. Other granules showed a maximal absorption either at about 450 or 560 nm. The maximum at 450 nm is suggested to be caused by xanthommatin and that at 560 nm by ommines. The pigment screen inHeliconius is concluded to be a neutral grey filter.This work was supported by the Swedish Medical Research Council, grant No. B71-14X-104 06B and 070, Sällskapet för Medicinsk Porskning and Reservationsanslaget, and the Deutsche Forschungsgemeinschaft.     

Electron microscopic investigation of adrenergic and non-adrenergic axons in the rabbit SA-node

Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO₄ which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.This work was supported by grants from Åhlén-Stiftelsen, the Faculty of Medicine, University of Lund, Lund, Sweden, the United States Public Health Service (project 06701-04) and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05).     

Effect of colchicine on axonal transport and morphology of retinal ganglion cells

Summary Intraocular injection of colchicine in doses which do not affect the protein synthesis in the retina has profound effects on the axonal transport of protein in the retinal ganglion cells of the rabbit. Rapid axonal transport in these cells is completely inhibited after treatment with relatively low amounts of colchicine. In contrast to this, a certain fraction of the slow axonal transport is resistant to colchicine treatment. Colchicine in doses which completely inhibits fast axonal transport caused discrete morphological changes in the perikaryon and in the axon of the retinal ganglion cell. No disappearance of microtubules and no general proliferation of neurofilaments was observed in the perikaryon of the retinal ganglion cells. There was a slight or moderate increase in the number of filaments in the intra-retinal part of the axons of the retinal ganglion cells.This work has been supported by grants from the Swedish Medical Research Council (B71-12X-2543-03, B71-13X-2226-05A) and the Swedish National Cancer Society (265-B70-02X).     

Ultrastructure of blastocyst attachment in the mouse

Summary Mouse embryos have been examined with light and electron microscopy after fixation by perfusion with glutar aldehyde, and embedding in plastic.The Zona pellucida is dissolved gradually around the blastocyst just prior to attachment, and Zona free blastocysts exist only for a very short time.Blastocyst attachment is established when the trophoblast and uterine cell surface membranes lie within 150 Å apart over wide areas. The uterine epithelium does not show any signs of degeneration.Trophoblast attachment probably precedes decidual cell reaction.This work was supported by the Swedish Government Funds for Supporting Medical Research and the Swedish Medical Research Council (Project No. 12 X-70-02).     

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).