B. Sporrong

Cholinergic Mechanisms in Pial Vessels

SummaryPlexuses of cholinergic nerve terminals were demonstrated (acetylcholinesterase staining) in pial arteries (down to a diameter of about 15μ) at the base of the brain and on the brain convexities of mice, rats, rabbits, hamsters, guinea-pigs, and cats. The pial veins were less well supplied than the arteries. Consecutive formaldehyde gas treatment (to visualize adrenergic nerves) and acetylcholinesterase staining revealed that the adrenergic and cholinergic plexuses followed each other closely, the axon terminals running together in the same Schwann cell strands. This was confirmed by electron microscopy after KMnO4 fixation or 5-hydroxydopamine treatment. The varicosities of cholinergic and adrenergic axons were sometimes seen as close as 250 Å. In the neuro-effector area, the terminals of both nerve types (naked or surrounded by an incomplete Schwann cell covering) approached the smooth muscle cells as close as 800–1100 Å, and they were separated from the latter only by the fused neuronal and muscular basement membranes. In this area axo-axonal contacts were observed. The adrenergic, but not the cholinergic, nerves disappeared after bilateral removal of the superior cervical sympathetic ganglia. Isolated cat middle cerebral artery contracted strongly with acetylcholine, and the effect was inhibited by atropine.With regard to the cholinergic neural control of the intracranial arteries, it may have particular functional implications: (1) that these vessels do have a cholinergic parasympathetic innervation in contrast to most other vascular systems, for example, in the mesenterium, (2) that this cholinergic nerve supply was found to be about equally prominent as the adrenergic (sympathetic) innervation which, in some pial vessels, is even better developed than in the mesenteric arteries, and (3) that the adrenergic and cholinergic systems in the intracranial arteries may interact, even at the level of the neuro-muscular contacts, a complex situation which may be partly responsible for the previous difficulties in defining the autonomic neural influence on the brain circulation.

Ultrastructure of the autonomic innervation apparatus in the main pial arteries of rats and cats

The autonomic nerve supply, particularly its terminal portion, was studied by electron microscopy of the anterior and middle cerebral arteries of rats and cats. In agreement with previous histochemical information, the arteries received a considerable number of non-myelinated axons that were enclosed in a Schwann cell plasmodium. Close to the vascular wall, varicosed terminals were found together with smooth preterminal fibres. In KMnO4-fixed material from untreated animals, about half of the terminals contained an abundance of dense-cored, 50 nm synaptic vesicles, whereas the synaptic vesicles in the remainder of the terminals were empty. In terminals of both types, a smaller number of larger electron-dense vesicles of 100 nm was present. After treatment of the animals with 5-hydroxydopamine followed by glutaraldehyde and osmium fixation, dense-cored vesicles with a diameter of 50 nm up to 80 nm appeared in about the same number of terminals as were previously found to contain the electron-dense synaptic vesicles. The rest of the axon terminals remained unchanged. It is concluded that the terminals with dense-cored vesicles belonged to adrenergic nerves, whereas those with the empty 50 nm vesicles were cholinergic. Near the vessel, the Schwann cell sheath of the terminals was usually incomplete, and varicosities of either the same or different (i.e., adrenergic/cholinergic) type came in close apposition to each other with a distance between adjacent membranes of approximately 25 nm. This arrangement offers a structural possibility for an interaction mechanism between the two types of axon terminals. In the adventitia the terminals had either an incomplete Schwann cell sheath, or were completely naked. Varicosities of the terminals of both types of axons approached the outer muscular layer of the media with a distance of about 100 nm. In this neuro-effector area, axo-axonal appositions were also found. The pial arteries possessed a neuro-effector apparatus that fulfilled the criteria of a true autonomic innervation in the same way as with arterial vessels in various peripheral organs.

Amine mechanisms in enterochromaffin and enterochromaffin-like cells of gastric mucosa in various mammals.

SummaryThe stomach wall from a variety of mammals (mouse, rat, hamster, guinea-pig, rabbit, cat, dog, pig, monkey and man) was analyzed histochemically and chemically for the presence of histamine, arylethylamines and the corresponding amino acid decarboxylases.1.Apart from adrenergic nerve terminals, the major cellular stores for gastric amines were mast cells, enterochromaffin cells, and a system of morphologically similar cells designated as enterochromaffin-like cells.2.Using the histochemical o-phthaldialdehyde technique, histamine could be visualized in gastric mast cells from all species. In addition, a yellow formaldehyde-induced fluorescence indicating the presence of 5-hydroxytryptamine or some other indole, was found in the gastric mast cells from mice, rats, rabbits, cats, dogs, pigs and monkeys. In hamsters the mast cells exhibited a green colour rather indicating the presence of a catecholamine.3.All argyrophilic epithelial cells in the stomach mucosa emitting a formaldehyde-induced fluorescence were classified as enterochromaffin. Yellow-fluorescent, presumably 5-hydroxytryptamine-containing, enterochromaffin cells were recognized in all species. In addition, the stomach mucosa of rabbits and cats contained scattered green-fluorescent enterochromaffin cells; in microspectrofluorometric analysis the fluorophore was identified with the formaldehyde condensation product of dopamine. The presence of dopamine in gastric mucosa of these two species was further confirmed by chemical estimation in combination with thin-layer chromatography.4.Already the presence of different arylethylamines in the enterochromaffin cells indicates the existence of several distinct cell types. A further classification was made on the basis of a different sensitivity to the amine-depleting action of reserpine.5.The enterochromaffin-like cells are argyrophil but not argentaffin. A distinguishing feature of these cells is their ability to produce and store arylethylamines, such as dopamine, upon administration of the amine precursor (e.g.l-DOPA), although normally the cells are devoid of fluorogenic amines demonstrable with the formaldehyde method. In addition, the enterochromaffin-like cells of mouse and rat store histamine histochemically detectable with the o-phthaldialdehyde technique. In the mouse and rat these cells appear to be the major site of gastric histidine decarboxylase. In no other species could histamine or histidine decarboxylase be demonstrated in the enterochromaffin-like cells.6.The regional distribution of the enterochromaffin and enterochromaffin-like cells varied markedly, but the localization of these two cell systems was characteristic for each species. It was very noticeable that the distribution of the cells within the gastric mucosa often was not limited to one or the other of the histologically definable glandular areas.7.From observations on the regional and topographic distribution of DOPA decarboxylase (i.e. non-specific l-amino acid decarboxylase) it is suggested that the enzyme occurs in both enterochromaffin and enterochromaffin-like cells, in particularly high concentration in the former cell type.8.The enterochromaffin and enterochromaffin-like cells — i.e. the entire system of gastric argyrophil cells — may have an endocrine function. It is evident that a thorough knowledge of the properties and distribution of the many types of cells comprising this system will assist in defining their respective endocrine mechanisms.

Adrenergic and cholinesterase-containing neurons of the heart

SummaryThe 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.

Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

SummaryIn the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.

Comparative study of the autonomic innervation of the mammalian ovary, with particular regard to the follicular system

SummaryThe autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections.

SummaryThe argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.

Cellular stores of histamine and monoamines in the dog stomach

Abstract The gastric mucosa of the dog is rich in histamine. Fluorescence microscopic analysis showed that in this species gastric histamine is stored in mast cells, which differ from mast cells at other sites in that they are resistant to the histamine-releasing action of Compound 48/80. Previously, it has been established that in the rat the major portion of gastric histamine is stored in a specific, enterochromaffin-like epithelial cell system in the oxyntic gland area; in contrast to the enterochromaffin cells of the pyloric region they contain no 5-hydroxytryptamine. A morphologically similar cell system could be demonstrated in the antral region of the dog stomach. In this species, however, these enterochromaffin-like cells are devoid of histamine, as well as of histochemically detectable monoamines. Nevertheless, they were found capable of producing and storing monoamines from exogenous precursor amino acids. In the rat, gastric histamine is probably produced locally since the enterochromaffin-like cells are rich in histidine decarboxylase. Histidine decarboxylase activity in the dog stomach was found to be low, which contrasts with the high gastric histamine content. The origin of gastric histamine in this species is still unknown. It is possible that most of it results from an active uptake of preformed, circulating histamine rather than from local biosynthesis.

Pregnancy is associated with extensive adrenergic nerve degeneration in the uterus. An electronmicroscopic study in the guinea-pig

Abstract The uterus of virgin guinea-pigs is supplied with a well-developed system of adrenergic nerves, characterized by 50 nm diameter synaptic vesicles whose electron-density is enhanced following administration of 5-hydroxydopamine. In the course of pregnancy, an increasing number of nerves with signs of various stages of degeneration are seen, varying from a slight condensation of the axonal content or myelin figures, to highly osmiophilic dense bodies lacking plasma membrane and with no organelles discernible. Incipient degeneration in a small number of axons is evident already in early pregnancy (up to 10 days post coitum ). At late pregnancy (40–50 days post coitum ), almost all adrenergic nerve terminals have disappeared as a consequence of degeneration, so that only a few isolated heavily degenerated axon terminals remain visible. Neither normal nor degenerating axons are found in the myometrium at full term. The degenerative changes occur earlier and are, at the various pregnancy stages, more pronounced in that part of the uterus surrounding and expanded by the fetus, compared to those segments of the uterine wall separating the fetuses. Thus, the normal neuromuscular relationship in the uterus during such an entirely physiological condition as pregnancy is lost entirely, in contrast to other sympathetically innervated organs, where the nerve plexus usually keeps pace with volume changes in the effector tissue.

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

SummaryIn the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.