Leena Rechardt

Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

SynopsisPineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.

Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain.

The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture. Direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity were used to identify the capillary endothelial cells.

Reinnervation of Human Skin Grafts: A Histochemical Study

The reinnervation of nine human skin grafts was investigated using histochemical thiocholine methods for the demonstration of cholinesterases. The regenerated cutaneous nerves showed both specific acetylcholinesterase and nonspecific cholinesterase reactions. In the youngest specimens, taken 3 weeks after the grafting, such regenerated nerves were seen both at the subdermal level under the graft and at the margins of the graft. These nerves seemed to orient toward the denervated graft area. The growing nerves were generally distributed in a random fashion. The reinnervation of some hair follicles, erector pili muscles, and sweat glands were observed in well-innervated full-thickness and thick partial-thickness skin grafts. It is suggested that this target-organ control of regenerating nerves occurs as a result of the action of chemotactic factors. A well-innervated graft bed seems to be important for optimum reinnervation of skin grafts. Fibrosis and scarring seem to hamper nerve regeneration.

Electron microscopic demonstration of cholinesterases in nervous tissue

SummaryAcetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rats spinal cord using the Karnovsky-Roots modification of Koelles thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.

Differentiation of neuroblastoma cells induced by nerve growth factor in vitro

Durch Züchtung in vitro wird gezeigt, dass Zellen eines menschlichen Neuroblastoms unter dem Einfluss eines Nervenwachstumsfaktors die Fähigkeit haben, sich in Nervenzellen zu differenzieren und somit die Zellen dieses bösartigen Tumors histogenetische Potenzen besitzen, welche nicht aktiviert werden.

Electron microscopical demonstration of adenylate cyclase activity in nervous tissue.

SummaryAdenylate cyclase (EC 4.6.1.1) activity stimulated by norepinephrine and dopamine was demonstrated histochemically by electron microscopy in the cerebral cortex and caudate nucleus of the rat. The precipitating agent in the histochemical reaction was cobalt, which was shown biochemically to increase the adenylate cyclase activity. The reaction product was located in the synapses, being contiguous attached to the postsynaptic membrane. It was also located in the plasma membrane of some nerve fibers. Alloxan, the specific inhibitor of adenylate cyclase, inhibited the reaction in the cerebral cortex and caudate nucleus, and haloperidol had a somewhat similar effect in the caudate region.

Catecholamine-synthesizing enzymes in the rat pituitary

SummaryThe catecholamine-containing nerve fibers of the rat pituitary were studied by immunohistochemical demonstration of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Immunohistochemical demonstration of TH confirms earlier catecholamine fluorescence histochemical studies showing a fine network of varicose fibers in both the intermediate and the neural lobe, with the most dense aggregation of fibers at the border between the lobes. DBH-immunoreactive fibers were much less in number, and confined to the neural lobe, where both vascular and parenchymal fibers were seen. With the antibody to PNMT bright staining was seen in all the glandular cells of the intermediate lobe, while the neural lobe was negative. No immunoreactive structures were observed in the anterior lobe.Functionally the study confirms the presence of an extensive dopaminergic innervation of the neurointermediate lobe, giving an anatomical basis for the tonic inhibitory action of dopamine on the intermediate lobe cells and for recent observations attributing dopamine a local regulatory function also in the neural lobe. In addition to vascular noradrenaline-containing fibers as described earlier the study shows parenchymal DBH-immunoreactive fibers in the neural lobe, suggesting a local role for noradrenaline in this lobe. The nature of the cellular PNMT-immunoreactivity in the intermediate lobe remains to be established. The cellular localization of the PNMT-immunoreactivity was distinctly different than that of the α-MSH-immunoreactivity within the intermediate lobe cells and reserpine treatment did not affect the PNMT-immunoreactivity, although it induced a heterogenous depletion of α-MSH and related peptides. Cross-reaction of the PNMT-antibody with the known secretory products of these cells thus seems unlikely. Biochemical studies are needed in order to show whether an actual PNMT activity is present.

Observations on the morphology and histochemistry of the rat neostriatum in tissue culture

Abstract Cells from the neostriatum of newborn rats were cultured for 30 days in vitro . The morphology of the cultures was studied using light and phase contrast microscopy and fluorescence microscopy. The studies were completed by demonstration of monoamine-oxidase, acetylcholinesterase and alkaline phosphatase enzyme activities. During the culture period cells possessing the characteristics of mature neurons were observed. Most neurons were small to medium-sized bipolar or tripolar cells but large nerve cells were few in number. Some neurons showed a distinct acetylcholinesterase activity in the cytoplasm. Monoamine-oxidase activity was observed in both neurons and non-neural cells. Alkaline phosphatase activity was present on the surface of some flat cells, although many similar cells were devoid of activity. No catecholamine fluorescence was seen in the cultured cells but neurons as well as glial cells and flat non-neural cells took up l -DOPA. Some cells also took up dopamine at low concentrations. Several different transmitter mechanisms are involved in the neuronal organization of the neostriatum and its related regions so that its morphology is difficult to define. However, the present work shows that tissue culture is a possible approach to the study of the instrinsic neuronal organization of the neostriatum.

Histochemically demonstrable catecholamines and cholinesterases in nerve fibres of rat dorsal skin

SummaryHistochemically demonstrable cholinesterases of rat skin and cutaneous nerves hydrolyze acetylthiocholine iodide and butyrylthiocholine iodide. Cholinesterase activity of the skin was located in the epidermis, in the hair follicles at the level of the sebaceous glands, in adjacent parts of the sebaceous glands, in erector pili muscles and their nerves, in cutaneous and subcutaneous nerves and nerve trunks, including some nerves accompanying cutaneous blood vessels, and in the membranes of fat cells. No encapsulated nerve endings were found. In the nerves of erector pili muscles there was some neurilemmal non-specific cholinesterase activity, demonstrated in the presence of 10−5 M BW 284C 51, and specific acetylcholinesterase activity resistant to 10−5 M iso-OMPA. The cholinesterase activity in other cutaneous nerves was inhibited by 10−5 M iso-OMPA but was resistant to 10−5 M BW284 C 51, thus representing mainly non-specifc cholinesterase (nsChE) activity.The adrenergic nerves of the dorsal skin, as revealed by glyoxylic acid-induced fluorescence (GIF), were located in association with erector pili muscles and surrounded arteries and arterioles. Small fluorescent nerves were situated in subcutaneous nsChE-positive nerve trunks.Using GIF and cholinesterase techniques performed either simultaneously or consecutively, it was found that the nsChE-positive, probably sensory, nerves accompanying blood vessels were fewer in number than the fluorescent adrenergic nerves and ran a course independent of them. No cholinesterase reaction was seen in the fluorescent adrenergic nerves when short incubation times were used. When the incubation time was prolonged overnight, the nsChE reaction closely followed the course of fluorescent adrenergic nerves.

Light and electron microscopic demonstration of cholinesterases of the cultured sympathetic ganglia of the chick embryo

SummaryCholinesterases were demonstrated light and electron microscopically completed with fluorescence microscopic studies in the cultured sympathetic ganglion cells of chick embryos.The sympathetic neurons were cultured for various periods in organotypic cultures with Nerve Growth Factor. The cholinesterase reactions were performed according to the Koelle-Gomori, Karnovsky-Roots and Lewis-Shute modifications. Successive demonstrations of amine fluorescence and cholinesterases were carried out.Variable intensity of enzymatic activity was found in the nuclear and cytoplasmic membranes, in the endoplasmic reticulum and in the Golgi apparatus. Nerve fibers were also surrounded with the reaction product.In the specimens processed according to the Karnovsky-Roots modification, which gave a good ultrastructural preservation, reaction product was observed in addition to the neurons also in the cytoplasmic membranes of the cells containing large dense cored vesicles and in the outer membranes of the fibres containing small dense cored vesicles.It is concluded that the cells populating the sympathetic ganglion of chick embryo maintain acetylcholinesterase activity in the organotypic culture without preganglionic innervation or other exogenous stimulation.