The major ganglion in the pelvic plexus of the male rat

Summary To further evaluate the role of autonomic ganglia in the regulation of pelvic visceral activity, the neural elements in the major pelvic ganglion of the male rat have been studied with histochemical and electron microscopic techniques. The principal findings are that the ganglion is composed of cholinergic and adrenergic ganglion cells as well as small intensely fluorescent (SIF) cells. Polarity in the ganglion is indicated by clustering of small ganglion cells which stain intensely for acetylcholinesterase (AChE) along the pelvic nerve while larger cells, with weak to moderate AChE activity, collect near small branches of the hypogastric nerve. Some cholinergic ganglion cells are enclosed by a plexus of adrenergic terminals. SIF cells appear to be in contact with both cholinergic and adrenergic cells, although many of the fluorescent beads around adrenergic neurons may be short dendrites of ganglion cells, rather than processes of SIF cells. Two types of SIF cells may be distinguished on the basis of size and morphology of their granulated vesicles. Afferent synapses of the cholinergic type were common on SIF cells of the large granule and small granule type. Portions of SIF cells with large granules occur within the capsule of ganglion cells. Contacts seen here were interpreted as efferent synapses from SIF cells to the dendrites of ganglion cells.     

Autofluorescence in freshly isolated adult human liver sinusoidal cells

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of exogenous fluorophores, especially when labelling of live cells where the quencher is not compatible.Key words: Autofluorescence, endogenous fluorophores, liver, endothelial cells, lipofuscin     

Catecholamine-rich cells and varicosities in bovine splenic nerve,vesicle contents and evidence for exocytosis

The bovine splenic nerve trunk contins mast cells, ganglion cells, small intensely flurescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structure could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300–800nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120–140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45–55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicle in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.

The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Estimation of fluorescence lifetime of lipofuscin fluorophores contained in lipofuscin granules of retinal pigment epithelium of human cadaver eyes without signs of pathology

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE—fundus autofluorescence (FAF).     

Age-related changes in the amount and intensity of the fluorescence of small, intensely fluorescing cells in the autonomic ganglia of rats

The number and intensity of fluorescence of small, intensely fluorescent cells were measured on serial slices of main pelvic (MPG) ganglion and lumbar ganglia of sympathetic trunk (LG), treated by modified Falck method, on days 1, 7, 14, 28, and 26-30 months of age. The content of paraform-induced fluorescence increased with age of two weeks and later in SIF cells of MPG and LG. The number of SIF cells in LG decreased with age, while that of MPG increased. The growth of a number of SIF cells in MPG was detected in large clusters.     

Acridine orange distribution and fluorescence spectra in myoblasts and single muscle fibers

Acridine orange (AO) fluorescence spectra in nuclei and cytoplasm of living myoblasts L6J1 and frog single muscle fibers have been studied using spectral scanning system of Leica TCS SL confocal microscope. AO fluorescence spectra in salt solutions dependent on free AO concentrations or in complex with DNA have also been obtained. Myoblast nuclei fluoresced in the green spectral region with maximum at about 530 nm; nucleoli had the brightest fluorescence. The fluorescence of nuclear chromatin was not uniform. Similar fluorescence of nuclei and nucleoli was observed in frog single muscle fibers. Uniform, weak, green fluorescence was observed in the myoblast cytoplasm. In the sarcoplasm of muscle fibers, AO green fluorescence was seen in A discs. In the cytoplasm of myoblasts and muscle fibers stained with AO, different red, yellow, and green fluorescent granules, which were acidic organelles, were visualized. The comparison of AO fluorescence spectra in living cells with AO fluorescence spectra in buffer solutions with different AO concentrations and AO in complex with DNA enables the estimation of the AO concentration in acidic granules. It is important for the evaluation of these cellular organelles functions in intracellular transport, adaptation, and apoptosis, as well as in a number of pathological processes.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Soluble lipofuscin in commercially-available human serum albumin solutions

Two studied commercial human serum albumin solutions had developed yellow colors during storage. These yellow materials were isolated and shown to be soluble lipofuscin. Aqueous solutions of this lipofuscin exhibited fluorescence spectra with 355 nm excitation and 432 nm emission maxima. After acid hydrolysis of this lipofuscin a nonhydrolysable lipid-melanin fraction was obtained. Ethanol-ether extraction yielded a lipid-containing solution. When evaporated and mixed with water, a solution-suspension was obtained that produced very similar fluorescence spectra to those described above, with 368 nm excitation and 432 nm emission maxima. The separated melanin component was not fluorescent. The isolated lipofuscin exhibited a weak electron paramagnetic resonance spectrum and its g-value has been found to be 2.0069 and its line width 9.8 G. The albumin solution contained approximately 0.23 g of melanin precipitate per 9.31 g of soluble lipofuscin isolated from 25 g of albumin. The deleterious cardiac, pulmonary, renal and clotting changes associated with the use of albumin solution might be due to this lipofuscin.     

Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic,neurofilament immunoreactive myenteric neuron types of human small intestine

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO₄ or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO₄ or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.     

Principal neurons projecting to the pineal gland in close association with small intensely fluorescent cells in the superior cervical ganglion of rats

Summary The localization in the superior cervical ganglia (SCG) of small, intensely fluorescent (SIF) cells and of principal nerve (PN) cells innervating the pineal gland was examined in adult male Sprague-Dawley rats. PN cells were demonstrated by means of the retrograde neuron-tracing method using the fluorescent tracer Fluoro-Gold (FG) injected into the pineal gland. SIF cells were visualized by the formaldehyde-induced fluorescence method. Twentynine percent of the FG-labeled PN cells were found closely associated with SIF cells. In the rostral half of the ganglion, 43% of the SIF cells were situated in juxtaposition to one or several labeled neurons. The possible influence of SIF cells on the regulation of pineal metabolism is discussed with respect to their role as both local endocrine cells and interneurons.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

The autofluorescent products of lipid peroxidation may not be lipofuscin-like

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Synaptic contacts between SIF cells immunoreactive to cholecystokinin (CCK-8) in inferior mesenteric ganglia in the guinea-pig

Cell bodies and processes of SIF cells were found to be CCK-like immunoreactive within the mesenteric ganglion of the guinea-pig. Immunoreactivity was contained in granular vesicles, the labeling being chiefly associated with granules, about 100 nm in diameter. Asymmetric synaptic contacts between CCK-like immunoreactive SIF cells have been identified and a possible functional implication regarding intraganglionic connections for these SIF cells is discussed.     

Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.     

Photobleaching and Fluorescence Recovery of RPE Bisretinoids

The autofluorescence of the retina that originates primarily from lipofuscin fluorophores in retinal pigment epithelial cells, is observed to undergo photobleaching during the acquisition of fundus autofluorescence images. Bisretinoid fluorophores isolated from retinal pigment epithelial cells have the spectral characteristics consistent with their being the source of fundus autofluorescence. Clinically relevant experiments were designed to better understand conditions in the micromilieu of bisretinoid fluorophores that can influence fluorescence efficiencies, photobleaching, and subsequent fluorescence recovery of this fluorophore. The consumption of the bisretinoid A2E due to photooxidation-induced degradation was quantified in solvent systems of variable relative permittivity (formerly called dielectric constant), in micelles, and in phospholipid vesicles of varying composition. Reorganization within biphasic systems was also examined. A2E content was measured by high performance liquid chromatography (HPLC) and fluorescence intensity was quantified spectroscopically. As solvent polarity was increased, A2E fluorescent spectra exhibited red-shifted maxima and reduced intensity. A2E was depleted by light irradiation and the loss was more pronounced in less polar solvents, lower concentrations of anionic surfactant, and in gel- versus fluid-ordered phospholipid liposomes. Conditions that permit A2E aggregation promoted photooxidation/photodegradation, while movement of A2E between bisphasic systems was associated with fluorescence recovery after photobleaching. The fluorescence characteristics of A2E are subject to environmental modulation. Photooxidation and photodegradation of bisretinoid can account for fundus autofluorescence photobleaching. Return of fluorescence intensity after photobleaching likely occurs due to redistribution of A2E fractions amongst co-existing heterogeneous microdomains of the lysosomal compartment.     

Intraganglionic portal sinus located between small intensely fluorescent (SIF) cells and principal ganglionic neurons in the inferior mesenteric ganglion of the guinea pig

Summary The vascular system in the inferior mesenteric ganglion of the guinea pig was studied to clarify the transport pathway of transmitters released by the small intensely fluorescent (SIF) cells to the principal ganglionic neurons. Reconstruction of about 1500 1-m-thick serial sections of the ganglion demonstrated its portal system. SIF cells were tightly packed and formed two or three clusters under the capsule of the ganglion. Branches from the inferior mesenteric artery ran directly toward these clusters and broke up into a number of coiled and looped sinusoid capillaries among the SIF cells. They then drained into a large sinus surrounding the clusters in the ganglion. Capillaries were derived from this sinus and ramified among the principal ganglionic neurons. After supplying the neurons, these vessels drained into veins surrounding the ganglion. Therefore, as we observed two distinct groups of capillaries, we call this sinus the intraganglionic portal sinus. All the transmitters secreted from the SIF cells are collected into this intraganglionic portal sinus and are then conveyed through the capillaries to the principal ganglionic neurons.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p &lt; 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.