Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization

Summary An attempt was made to study the subcellular localization of calcium in carotid body glomus cells of adult rats using fixation with glutaraldehyde followed by treatment with a mixture of pyroantimonate and osmium tetroxide. Precipitates were seen as electron-dense particles (EDP) in the glomus cells, mostly within membrane-bound organelles, such as dense-cored vesicles, mitochondria, small clear vesicles, multivesicular bodies, and especially in lysosomes. However, EDP were also seen in the nuclei and in the free cytoplasm of the glomus cells and even outside them.Preincubation of carotid bodies in media containing calcium and either high potassium or calcium-ionophore A 23187 resulted in a marked increase in the general precipitation pattern, there being an increased amount of EDP both in the glomus cell nuclei and in the cytoplasm. Dense-cored vesicles more often showed precipitates than those in the controls. Some dense-cored vesicles contained multiple precipitates, typically located in the electron-lucent area between core and vesicle membrane.Extensive diffusion of ions probably occurred during fixation before precipitation, making the localization of calcium and other precipitating cations unreliable. However, it is possible that precipitates, which were regularly seen in the dense-cored vesicles, may reflect the content of bound calcium. The possible significance of calcium in glomus cell function is discussed, and the need for more adequate methods is emphasized.The present study has been supported by grants from the Finska Läkaresällskapet and the Sigrid Jusélius Foundation, Helsinki, FinlandWe wish to express our gratitude to Dr. Robert Hamill of Eli Lilly Co. for kindly providing us with the ionophore A 23187. Technical assistance by Mrs. S. Huhtaniitty and Mrs. T. Stjernvall is gratefully acknowledged     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.     

Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.     

THE EFFECTS OF RESERPINE AND HYPOXIA ON THE AMINE-STORING GRANULES OF THE HAMSTER CAROTID BODY

The carotid bodies from control, reserpine-treated, and hypoxia-treated hamsters were fixed with phosphate-buffered glutaraldehyde and osmium tetroxide, s-Collidine-buffered osmium tetroxide, or phosphate-buffered glutaraldehyde followed by potassium dichromate incubation. Following glutaraldehyde-osmium tetroxide fixation no differences in density or population of the electron-opaque granules in the glomus cells of either control or experimental animals were observed. With s-Collidine-buffered osmium tetroxide and the glutaraldehyde-dichromate technique a marked decrease in density without an appreciable reduction in number of granules was noted after reserpine treatment, while in hypoxia-treated hamsters the density and population of the granules were not different from those of the controls. The results indicate that reserpine depletes the amines without granule disappearance and that hypoxia does not affect the amine content of the granules. It is suggested that following glutaraldehyde-osmium tetroxide double fixation, persistence of the density of the granules in reserpine-treated animals is due primarily to the nonamine content, and that the amines in the glomus cells are probably not directly involved in the respiratory reflex.     

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture

Synopsis Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeksin vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor.In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlierin vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensely fluorescent (SIF) cells were observed.Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.     

Calbindin D-28k immunoreactive nerve fibers in the carotid body of normoxic and chronically hypoxic rats

The distribution and ultrastructural characteristics of calbindin D-28k immunoreactive nerve fibers were examined in the carotid body of the normoxic control rats by light and electron microscopy, and the abundance of calbindin D-28k fibers in the carotid body was compared in normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). Calbindin D-28k immunoreactivity was recognized in nerve fibers within the carotid body. Calbindin D-28k immunoreactive nerve fibers appeared as thin processes with many varicosities. They were distributed around clusters of glomus cells, and around blood vessels. Immunoelectron microscopy revealed that the calbindin D-28k immunoreactive nerve terminals are in close apposition with the glomus cells, and membrane specialization is visible in some terminals. Some dense-cored vesicles in the glomus cells were aggregated in this contact region. The chronically hypoxic carotid bodies were found to be enlarged several fold, and a relative abundance of calbindin D-28k fibers was lesser than in the normoxic carotid bodies. When expressed by the density of varicosities per unit area of the parenchyma, the density of calbindin D-28k fibers associated with the glomus cells in chronically hypoxic carotid bodies was decreased by 70%. These immunohistochemical findings indicate a morphological basis for involvement of calcium binding protein in the neural pathway that modulates carotid body chemoreception.     

Studies of Osmium Deposits in the Carotid Body of the Cat

Double aldehyde fixed carotid bodies and small pieces of vagus nerve of cats were incubated in 3 mM copper sulfate and 0.5 mM potassium ferricyanide in 0.05 M acetate buffer (pH 5.6) for 30 minutes at room temperature. Several modifications of this procedure were also attempted. Tissues were then postosmicated with 2% unbuffered osmium tetroxide and heated to 50-55 C for ten minutes. Under the electron microscope carotid body cells exhibited fine osmium deposits within cisternae of endoplasmic reticulum, saccule and vesicles of Golgi complex, and cristae of mitochondria. Intense osmium precipitation was also noted in the mitochondria of nerve endings. In addition, much more intense, more conspicuous and more localized reaction wan noted in the intraperiod lines of the myelin sheath of nerves. Deposits here were rod-shaped, displaying considerable variation in length. These results are discussed in the light of previous findings on osmium deposits in various tissues. It was concluded that the osmium reaction is unspecific, and that histochemical methods employing hot osmium tetroxide to amplify enzymatic activities may therefore not be reliable.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.     

Electron microscope studies of the human epidermis: the clear cell of Masson (dendritic cell or melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Electron Microscope Studies of the Human Epidermis The Clear Cell of Masson (Dendritic Cell or Melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Regulation of chemoreceptor sensitivity in the carotid body: the role of presynaptic sensory nerves

Several neural and vascular mechanisms regulate the sensitivity of carotid body chemoreceptors to hypoxia, hypercapnia, and acidosis. Factors that control blood flow and oxygen delivery in the carotid body along with those that augment or diminish catecholamine release from glomus cells can have major effects on chemoreceptor function. In addition, the sensory nerves themselves may participate in the regulation of chemoreceptor sensitivity. A portion of the carotid body's sensory nerves are presynaptic to glomus cells. In response to stimulation, the sensory nerve terminals exhibit ultrastructural changes that resemble changes associated with increased release of transmitter from motor nerves: 1) the number of small (synaptic) vesicles decreases; and 2) coated vesicles and coated regions of cisternal membrane increase in number during stimulation. If sensory nerves of the carotid body release a neurotransmitters, sensory nerve activity could influence glomus cell secretion of catecholamines or other substances tha modify chemoreceptor sensitivity. Such an effect could be produced in the carotid body by hypoxia and other conditions that stimulate the sensory nerves or it could result from antidromic activity evoked in the sensory nerves by primary afferent depolarization of their terminals in the CNS.     

Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves

Summary Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is elminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.     

Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves

Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is eliminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.     

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

Summary The relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.This work was supported by grants from the United States Public Health Service (06701-04), the Faculty of Medicine, University of Lund, Lund, Sweden and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05). Svenska sällskapet för medicinsk forskning.     

Fixation of proteins by osmium tetroxide potassium dichromate and potassium permanganate

Summary The crosslinking abilities of osmium tetroxide, potassium dichromate and potassium permanganate towards bovine serum albumin and bovine -globulin were investigated by chromatography with Sephadex G-200. Osmium tetroxide had a moderate crosslinking ability towards these proteins, the others had little or none. Chromatography with Sephadex G-50 permitted the oxidative cleavage of the proteins by these oxidative fixation agents to be studied. Potassium permanganate caused much fragmentation of the proteins and destruction of the tyrosine and tryptophan residues. Osmium tetroxide and potassium dichromate caused only a small amount of protein cleavage. These results were corroborated by polyacrylamide gel electrophoresis and viscosimetric studies. The significance of the results for tissue fixation is discussed.