Ch. Owman

Evidence for regenerative axon sprouting of central catecholamine neurons in the rat mesencephalon following electrolytic lesions

Abstract By means of histochemical fluorescence techniques, evidence has been obtained for regenerative sprouting of axons from catecholamine neurons in the rat mesencephalon 1–7 weeks after electrolytic destruction of the substantia nigra and part of the ventromedial midbrain tegmentum. Three days after the lesion, accumulations of catecholamine occurred in coarse, beaded and distorted fibers on the borders of the lesion within the ascending catecholamine fiber tracts. Such catecholamine accumulations in proximal portions of severed axons or axon collaterals are well known; these accumulations showed a progressive decrease after approximately 1 week. In the present study an additional phenomenon was discovered between 7 and 19 days after lesion, namely the appearance of numerous, densely packed, fine, varicose, fluorescent fibers, probably identical with sprouts from catecholamine neurons. These fibers had an abnormal distribution outlining the lesion border and the electrode tracks, sometimes coursing through the lesion and even invading the walls of blood vessels penetrating, or situated close to, the lesion. These numerous newly appearing fluorescent fibers, including those invading the blood vessel walls, were equally prominent in animals with their intracranial arteries sympathectomized by bilateral removal of the superior cervical ganglia. Hence, these fibers were of central origin. The fluorescence of the newly formed fibers was characteristic for catecholamines, and the presence of noradrenaline could be established by means of microspectrofluorometric analysis in fibers invading sympathectomized vascular walls. Although the catecholamine fluorescence in the abnormal fibers on the border of the lesion had decreased at 7 weeks, it persisted in the new fibers invading the vessel walls, which may thus constitute a terminal area of distribution for the regenerating central catecholamine neurons. The phenomenon of regenerative sprouting in the central nervous system has previously been demonstrated, but this is the first direct evidence of regeneration of central monoamine fibers.

Origin and distribution of VIP (Vasoactive Intestinal Polypeptide)-nerves in the genito-urinary tract

SummaryVIP (Vasoactive Intestinal Polypeptide)-immunoreactive nerves were found throughout the genito-urinary tract of the cat; they were less numerous in the guinea pig and in the rat. In the cat, VIP nerves were particularly numerous in the neck of the urinary bladder and proximal urethra, in the uterine cervix and in the prostate gland. The nerves were found in smooth muscle, around blood vessels and in the connective tissue immediately beneath the epithelium. Ganglia were found below the trigonum area of the bladder, in the wall of the proximal urethra, and in paracervical tissue. VIP-immunoreactive nerve cell bodies occurred in all these ganglionic formations. These ganglia probably represent the origin of the VIP nerves of the genital tract since their removal in the female cat greatly reduced the VIP nerve supply. Transection of the hypogastric nerves had no overt effect. Transection of the cervix eliminated the VIP nerves above the level of the lesion, except those in the ovaries, supporting the view that the VIP nerves of the uterus and the oviduct are derived from a paracervical source.

Regional distribution of mast cells containing histamine, dopamine, or 5‐hydroxytryptamine in the mammalian brain

Brain mast cells were studied in mice, rats, rabbits, hamsters, guinea pigs, cats, cows, monkeys, and humans with use of a variety of techniques. They were localized by staining with Astrablau or by toluidine blue-induced metachromasia and characterized by their ultrastructural appearance and by the presence of histochemically demonstrable histamine (o-phthaldialdehyde fluorescence method). The identity of the fluorophore was secured by microspectrofluorometry. Mast cells in brain usually had a perivascular localization but were also found scattered in the parenchyma. The regional variations in the number of mast cells agreed with the histamine concentration as measured fluorometrically. The variation was in the order leptomeninges > hypothalamus > cerebral cortex = mesencephalon > cerebellum = brain stem. In addition to histamine, murine mast cells stored serotonin, whereas bovine mast cells contained dopamine, visualized histochemically by the formaldehyde technique.

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.

Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain

SummaryThe pineal gland of the rat is located near the brain surface and is via a slender stalk connected to lamina intercalaris which constitutes a cell formation between the habenular and posterior commissures, continuing to the subcommissural organ. The stalk and lamina intercalaris, like the pineal proper, exhibited a yellow, formaldehyde-induced fluorescence which showed the histochemical and pharmacological properties of 5-HT. All these structures were richly supplied with catecholamine-fluorescent nerves which could be further followed rostrally from lamina intercalaris, mixing with the non-fluorescent commissural fibres and stria terminalis, into the medial habenular nucleus in which they extensively supplied both blood vessels and non-fluorescent nerve cells. Cytospectrofluorometric and chemical analysis suggested that the fluorescent nerves stored noradrenaline. This was supported by the finding that they disappeared after bilateral cervical sympathectomy (as did the fluorescent nerves in the pineal complex). In the medial habenular nucleus also catecholamine-containing and 5-HT-containing nerves of central origin were present.The occurrence of a rich, peripheral sympathetic innervation in the medial habenular nucleus of the brain offers possibilities for a previously not observed sympathetic influence on this nucleus. Also the arrangement, and the apparent continuity of the sympathetic innervation in the pineal gland, the lamina intercalaris, and the medial habenular nucleus, suggests some functional interconnection or coordination between these structures.

Histochemical and chemical studies on pre- and postnatal development of the different systems of “short” and “long” adrenergic neurons in peripheral organs of the rat

SummaryThe adrenergic innervation in the submaxillary gland, heart, kidney, small intestine, and accessory male genital organs and the development of the adrenal chromaffin cells and the sympathetic ganglia were studied in the rat from 15 days post coitum to 16 days post partum using the fluorescence histochemical method of Falck and Hillarp. The postnatal development of the noradrenaline concentrations in the heart and vas deferens was followed by fluorometric determinations.At about 15 days post coitum, the anlagen of the sympathetic chains were well visible in the form of two dorsal segmented columns of small branching sympathicoblasts exhibiting an intense catecholamine fluorescence. In the midline, ventrally to these two anlagen, another column of sympathicoblasts developed; this seemed to give rise to the prevertebral ganglia and to the short adrenergic neurons supplying the internal genital organs. At the level of the adrenal anlagen, small intensely fluorescent chromaffin cells were collected in two bilateral groups which became enclosed by adreno-cortical cells. This enclosure was, however, not complete even at two weeks post partum.Bundles of growing sympathetic nerves were visible in the periphery of the various organs studied at 19–21 days post coitum. A terminal innervation of the organs suggestive of a functional transmitter mechanism did not start to establish until at or immediately after birth. The final pattern of innervation was usually reached at about one week post partum, and the following development proceeded largely in the form of a quantitative increase in the number of nerves participating in the innervation apparatus. The adult level of noradrenaline in the heart and vas deferens was reached three to five weeks after birth. The small intestine was an exception in that the final pattern of innervation in the wall was attained immediately after birth.There was no overt difference in the rate of development of the terminal sympathetic innervation in organs supplied by short adrenergic neurons (accessory male genital organs) compared to the innervation of the submaxillary gland, heart and kidney, which receive classical long adrenergic neurons.

Properties of a new system of amine-storing cells in the gastric mucosa of the rat☆

Abstract Certain metabolic properties of a new system of amine-storing cells in the gastric mucosa of the rat have been investigated by combined histochemical and chemical methods for the demonsration of 3,4-dihydroxyphenylalanine (DOPA), dopamine, DOPA decarboxylase and monoamine oxidase. 1. 1. Three monoamine-containing cell systems occur in the stomach wall of normal, untreated animals: mast cells, adrenergic nerves, and enterochromaffin cells. 2. 2. Another large system of cells, structurally similar to the enterochromaffin cells, can be seen in the epithelium of the entire glandular portion of the stomach after administration of the amino acid precursor, L L-DOPA (or 5-hydroxytryptophan). 3. 3. These newly discovered “enterochromaffin-like” cells, which normally store neither catecholamines nor 5-hydroxytryptamine, can take up L-DOPA (or 5-hydroxytruptophan) by an active mechanism that can be blocked with cocaine. DOPA decarboxylase within the cells converts the amino acid to the corresponding amine (dopamine and 5-hydroxytryptamine, respectively), which is then retained in the cytoplasm by a reserpine-resistant storage mechanism. Monoamine oxidase is active in the breakdown of the amine formed. 4. 4. Also in the presence of D-DOPA - in vivo or in vitro - considerable quantities of dopamine can be demonstrated in the stomach wall, indicating the presence of racemizing enzymes in the stomach. 5. 5. The effect of some amino acid decarboxylase and MAO inhibitors (NSD 1015, NSD 1055, Ro 4-4602, pargyline, and nialamide) on gastric DOPA decarboxylase and monoamine oxidase has been studied.

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.