Heikki Hervonen

Effects of ageing on the histochemically demonstrable catecholamines and acetylcholinesterase of human sympathetic ganglia.

SummaryThe sympathetic ganglia of adult and aged humans were obtained during vascular, gynaecological and urological surgery, and studied using the formaldehyde-induced fluorescence (FIF) method for histochemical demonstration of catecholamines. Microspectrofluorimetry was applied to characterize the emission spectra of the fluorophores.The sympathetic ganglia contained two types of cells exhibiting FIF: the adrenergic neurons and the small, intensely fluorescent (SIF) cells. The adrenergic neurons contained pigment granules exhibiting autofluorescence with emission maximum at 530–540 nm. The number of these granules increased with age while the FIF of the neurons decreased. In the oldest patients, most of the neurons in the sympathetic ganglia showed only pigment fluorescence but not FIF. The decrease of the perikaryonal catecholamine stores is interpreted as a degenerative change leading to impaired function of the neurons. The patterns of AChE did not change with age.The SIF cells were sparse and were mostly observed near the ganglia, forming paraganglia of varying size. Because of the rarity of the SIF cells, the effect of ageing could not be judged reliably.

Fluorescence histochemical and electron microscopical observations on sympathetic ganglia of the chick embryo cultured with and without hydrocortisone

SummaryLumbar sympathetic ganglia of 12-day-old chick embryos were cultured in organ cultures for 14 days with 1, 10 or 100 mg/l of hydrocortisone or without it. Catecholamines were demonstrated by the formaldehyde-induced fluorescence method. For electron microscopy, the cultures were fixed with glutaraldehyde and osmium tetroxide.Two types of cells with catecholamine fluorescence were observed in the control cultures: (1) weakly fluorescent sympathetic neurons and sympathicoblasts with long nerve fibres, which were the most common cell type in the explant, and (2) brightly fluorescent cells with or without fluorescent processes, which were less common and were scattered in the explant.Hydrocortisone caused a great increase in the number of the brightly fluorescent cells. With 10 mg/l of hydrocortisone the increase was about ten-fold as compared with the control cultures. There was no change in the morphology of the cells, nor could any change be observed in the fluorescence intensity by eye.Electron microscopically the mature neurons were the most common cell type on the surface of the culture, while more immature sympathicoblasts were seen in the deeper layers. Cells were also found which contained large numbers of catecholamine-storing granular vesicles 105–275 nm in diameter. These cells were infrequent. They had round vesicular nuclei and resembled also in other respects sympathicoblasts or young nerve cells. One such cell was found in mitotic division by electŕon microscopy.Hydrocortisone caused a marked increase in the number of these granule-containing cells and their processes. Cells which could have been classified as the small intensely fluorescent cells of the mammalian ganglion type or their electron microscopic equivalent, the granule-containing cells were found neither in the control cultures nor in the hydrocortisone-containing cultures.It is concluded that most brightly fluorescent cells in cultured sympathetic ganglia of the chick are nerve cells or sympathicoblasts rich in amine-storing granular vesicles.

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.

Cytochemical demonstration of adenylate cyclase activity with cerium

SummaryCerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and choleratoxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.

Ultrastructural changes in the neurohypophysis of the aged male rat

SummaryThe neurohypophyses (neural lobes of the pituitary) of young adult (3–6 months) and aged (12–30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv % in the control animals to 1.1 Vv % in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.

Histochemical localization of adenylate cyclase in cultured sympathetic neurons

SummaryThe catecholamine-stimulated adenylate cyclase activity was histochemically demonstrated at ultrastructural level in the cultured neurons of the sympathetic ganglia of the chick embryo.Adenylate cyclase activity was demonstrated in the axolemma of the axons and axon terminals, however not inside the axons. Heavy precipitates of the enzymatic reaction product was observed intracellularly in some cell processes identified as dendrites of the sympathetic neurons.In contrast to the extensive reaction in the neuropil, the reaction for the catecholamine-stimulated adenylate cyclase was scarse in the perikarya of the neurons. Only occasional spots of adenylate cyclase activity could be demonstrated in the plasmalemma of some neurons, and no reaction product was observed in the cytoplasm.No difference in the localization of the adenylate cyclase activity was observed when either dopamine or noradrenaline were used for stimulation.The possible roles of the catecholamine-stimulated adenylate cyclase in the function of the interneuronal connnections between the aminergic neurons of the sympathetic ganglion are discussed.

Vacuolated neurons in the hypogastric ganglion of the rat

SummaryThe vacuolated neurons (VN) of the main hypogastric ganglion of the male rat were studied using the formaldehyde-induced fluorescence (FIF) method for the histochemical demonstration of catecholamines. Microspectrofluorimetry was performed to identify the fluorophores and to quantify the FIF. The thiocholine method (Koelle-Gomori) was used to demonstrate acetylcholinesterase activity. The fine structure of the VN was studied using glutaraldehyde/OsO4 fixation.(1) In the untreated adult male rat VN represent only a small population of the total number of hypogastric neurons (0.8–1.2%). The vacuoles are similar to those of the VN from the corresponding female ganglion. (2) The VN are considered to be adrenergic due to the nature of their fluorophore, indicating a primary catecholamine. (3) The first VN appear in the hypogastric ganglia at the age of 7 weeks. After testosterone administration to young rats, VN are found at the age of 4 weeks. (4) The basic fine structure of the VN is similar to that of other ordinary neurons of the hypogastric ganglia. (5) The content of the vacuoles could not be identified. (6) Indications of degeneration were not observed in the VN. (7) The VN are interpreted as being a functional stage of the “short” adrenergic neurons, which are under the control of steroid hormones. (8) Fifteen months after castration, no VN could be found in the hypogastric ganglia, while their number was normal in the corresponding control animals.

Axonal growth from the primitive sympathetic elements of human fetal adrenal medulla

Die primitiven sympathetischen Zellen aus fetalem Nebennierenmark des Menschen wurden in Gewebekultur studiert. Der neuronale Zellcharakter konnte durch bedeutendes Axonwachstum bestätigt werden.

Electron microscopic localization of adenylate cyclase activity of white and brown adipose tissue of the rat and chicken

SummaryBrown adipose tissue of newborn rats and chicken embryos and white adipose tissue of adult rats were studied. Adenylate cyclase (EC 4.6.1.1.) activity stimulated by 0.1 mmol/l noradrenaline was demonstrated using an electron microscopic histochemical method.The reaction product was visualized as a cobalt salt in the plasmalemma of the adipocytes. The adipocytes of the brown adipose tissue of the newborn rats showed most intense reaction in the outer surfaces of their plasmalemma. Alloxan totally inhibited the enzymatic reaction.The histochemical reaction used in the present study probably demonstrated the hormonal receptor sites in the plasmalemmas of the adipocytes which are stimulated by noradrenaline.

Ultrastructural changes in the neural lobe of the rat pituitary induced by reserpine treatment.

The doses of reserpine, which are generally used to deplete amines from the nervous tissue caused marked ultrastructural changes in the neural lobes of reserpine treated rats. The findings suggested depletion of neurosecretory granules, increased lysosomal activity and changes in the pituicytes.