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The localization of PKC-beta was studied in rat sympathetic neurons using a polyclonal antibody specific for the beta 1- and beta 2-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC-beta-LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC-beta-LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC-beta-immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC-beta-LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC-beta in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC-beta in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.

Sympathetic ganglion tissue of 3-months- and 18-months-old Fischer-344 rats was autotransplanted into the adrenal gland in order to determine the effect of aging on the survival of grafted neurons. Adrenal cortex was chosen as the host tissue because it is well vascularized and has a high concentration of glucocorticoids, which stimulate the synthesis of catecholamines. At 4 weeks following the transplantation, the density of neurons was decreased in all transplants, but approximately the same proportion of remaining neurons showed tyrosine hydroxylase immunoreactivity as in intact ganglia. At 8 weeks, a subpopulation of large neurons showed an increased accumulation of age pigment. The heavily pigmented neurons were usually devoid of catecholamines, whereas small non-pigmented neurons frequently showed strong catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. There was no marked difference between old and young animals in the survival of transplanted neurons. The results show that the sympathetic neurons from both 3-months-and 18-months-old animals survived the autotransplantation procedure. The humoral environment of the adrenal cortex may be beneficial for the restoration of the integrity of sympathetic neurons.

The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phorbol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.

Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%-80% of which were heavily labelled. About 5%-10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%-70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells. The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.

According to the earlier concept, the paraganglia of man are believed to degenerate during the first postnatal years after their dominance during the fetal period. Clinical case reports on persisting paraganglia led us to extensive exploration of surgical material obtained from urological and gynecological surgery. The formaldehyde induced fluorescence (FIF) was used for tracing the catecholamine containing tissues. The fluorescence intensities were recorded with a Leitz MPV 2 microspectrophotometer. Solitary, small paraganglia were found in all patients studied. They were especially frequent in the walls of the urinary bladder and in the connective tissue surrounding the urogenital organs. The intensity of the fluorescence was comparable to pharmacological standard of 10(-2) M noradrenaline and at the same level as the FIF of human fetal paraganglia. All cells of the paraganglionic clusters exhibited FIF and no signs of degeneration could be observed. It is suggested that the paraganglia of man do not degenerate postnatally but persist as a remarcable catecholamine reservoir, which might be of physiological importance.

Enkephalin-like immunoreactivity was demonstrated using the peroxide-antiperoxidase method. Only the adrenaline-storing cells of the hamster adrenal medulla were labelled.

Synaptosomes from one day old and adult rat brain were studied. Specific cytochemical methods for demonstrating monoaminergic (MA) nerve endings were used. Permanganate fixation after preincubation with 5-OHDA or alpha-methyl-NA demonstrated MA synaptosomes. Their number was small in the adult (less than 1 %) and still smaller in the one day old rat brain. The MA synaptosomes from developing rats were different from the adult ones. The large amount of endoplasmic reticulum in developing synaptosomes suggests that granular vesicles are formed from endoplasmic reticulum in nerve endings.

The development of the hypogastric ganglion of normal and testosterone-treated rats was studied using formaldehyde-induced fluorescence (FIF) method. The fluorescence intensities were recorded microspectrofluorimetrically. In normally developing rats cytoplasmic FIF decreases and cell size increases with age. In normally developing rats cytoplasmic FIF decreases and cell size increases with age. In testosterone-treated animals FIF increases during 2--6 weeks compared to the controls. The differences between control and experimental rats were significant. The diameters were significantly longer in treated animals in three and four week old groups. Vacuolated neurons were seen earlier in testosterone-treated rats. No changes in FIF or in cell size were noticed in the superior cervical ganglion. The male sex steroid, testosterone evidently influences the catecholamine turnover and cellular growth during development in the male pelvic ganglion.

The peroxidase-antiperoxidase method was applied to demonstrate enkephalin-like immunoreactivity in the human sympathetic ganglia. A network of immunoreactive fibres was found in light microscopy between the nonreactive neurons. Electron microscopy showed immunostaining to be restricted to axonal profiles containing both large and small vesicles. The strongest reaction was associated with large vesicles.

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.

The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.

The distribution of nerves containing immunoreactivity for the VIP and enkephalins has been demonstrated in the human prostate and seminal vesicles using the immunoperoxidase bridge. VIP-containing nerves were detected in both organs studied mainly in association with the epithelium, while nerves containing ELI seemed to be related to smooth muscle. Compared with the distribution of adrenergic and cholinergic nerves in the prostate marked differences in the density of the innervation were detected. The possible nature of these peptide-containing nerves is discussed.

The innervation of human lower respiratory tract was studied with special emphasis on airways with sodium-potassium glyoxylic acid (SPG) and acetylcholinesterase (AChE) methods to demonstrate catecholamine-containing and acetylcholinesterase-containing nerve fibers. AChE-method revealed a rich network of cholinesterase positive nerves both inside the bronchial glands where they run around and between the acini, and the airway smooth muscle from secondary bronchi to terminal bronchioli. No AChE-positive fibers were found in connection with the blood vessels or within the epithelium of bronchi or bonchioli. The AChE-positive nerve fibers in bronchial smooth muscle greatly outnumbered those containing catecholamine. The SPG-method revealed the presence of adrenergic nerves from the level of secondary bronchi to that of terminal bronchioli. These nerve fibers were most abundant in bronchial glands, where their amount was equal and distribution similar to those of AChE-containing nerve fibers. Outside the glands adrenergic fibers were constantly seen in connection with the bronchial blood vessels in connective tissues surrounding bronchi. A few nerve fibers were also present in airway smooth muscle from the secondary bronchi to terminal bronchioli.

A new combination of freeze-drying, formaldehyde induced fluorescence method for catecholamines and autoradiography of water soluble substances was developed and tested with different tissues containing biogenic amines. The freeze-dried tissue pieces were fixed with vaporours paraformaldehyde and OsO4 before embedding procedure. The effect of OsO4-vapour on the formaldehyde induced fluorescence was tested. The method produces semi-thin sections in which the fluorescence is well preserved and the tissue lightly stained with OsO4 which also fixes the steroid hormones used for radioautography.

The effect of ageing on SIF-cells was studied by the formaldehyde-induced fluorescence (FIF) method and by electron microscopy (EM). Microspectrofluorimetry was used to record emission spectra in FIF preparations. In newborn and in young adult (8--12 weeks) rats a single type of SIF-cell emitting greenish-yellow FIF was found while in aged rats a second type of SIF-cell emitting yellowish-brown granular FIF was also present. The intensity of the yellowish-brown FIF was lower than that of the greenish-yellow FIF. Also a few bright cytoplasmic fluorescent areas were occasionally found in some SIF-cells. The distribution of the SIF-cells through the ganglion did not change remarkably with age. In EM after glutaraldehyde-fixation in newborn and in young adult rats two types of small granule-containing (SGC) cells were distinguished according to the size of the dense cored vesicles, 1) 50--150 nm and 2) 50--250 nm. In aged rats, a third type of SGC-cells containing 50 x 250 nm elongated dense core vesicles could also be distinguished. After KMnO4-fixation in newborn and in young adult rats the classification was identical with glutaraldehyde-fixation. In aged rats three types of storage granules were found after KMnO4, 1) 100--300 nm empty vesicles and 2) 100--300 nm vesicles with small dense core, 3) 100--500 nm irregular in shape and filled with electron opaque material with a more dense core.

Three types of monoamine-storing cells were fixed with permanganates and analyzed ultrastructurally. The SIF cells contained the typical granulated vesicles characteristic of monoamines. On the other hand, both the catecholamine-storing cells in the adrenal medulla and the mast cells were devoid of a dense core in their monoamine vesicles. This is surprising, since permanganate is considered a reliable means of demonstrating monoamines at the fine structural level. The significance of this finding is discussed against the close relationship between SIF cells and cells of the adrenal medulla. Because permanganate as a fixative is widely used in monoamine fine structural cytochemistry, the present findings undoubtedly need further clarification.

The catecholamine content and morphology of the superior cervical and the hypogastric ganglion and the carotid body were studied in Spontaneously Hypertensive Rats (SHR) before (at the age of 6 weeks) and after (at the age of 20 weeks) becoming hypertensive, with Wistar Kyoto (WKY) rats as controls. The study was performed by formaldehyde-induced fluorescence method combined with quantitative microfluorimetry of catecholamines. At the age of 6 weeks the only significant difference observed between the rat strains was a greater number of small intensely fluorescent (SIF) cells in the superior cervical ganglion of SHR. At the age of 20 weeks the fluorescence intensity was higher in the principal neurons of the superior cervical ganglion and in glomus cells of the carotid body of SHR compared to WKY. The volumes of superior cervical ganglion and carotid body were larger in 20-week-old SHR compared to WKY. In the hypogastric ganglion differences were not found between SHR and WKY rats. The present results show differences in the superior cervical ganglion and in the carotid body of adult SHR compared to controls. These differences develop during the time period when the SHR become hypertensive, and might be functionally significant in the regulation or maintenance of the increased blood pressure in SHR rats.