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 rat pineal gland and vas deferens nerves

Summary In previous work of our laboratory it was demonstrated that collidine (2,4,6-trimethylpyridine) abolishes the core osmiophilia and chromaffin reaction from rat pineal gland and vas deferens nerves. This abolition was apparent when tissues were briefly incubated in collidine or when they were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer substance. These and other results strongly suggested that the histochemical effect of collidine was due to depletion of monoamines stored in the vesicles core. To examine this hypothesis we studied in this work the effect of collidine on tissues that have taken up tritiated noradrenaline. It was found that tritium was released very rapidly to the incubation medium when collidine was applied to fresh tissues. This effect was not observed with other commonly used buffers such as cacodylate or phosphate. It was also found that tritium release also occurred, although to a lesser extent, when tissues were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer, and this release was not significant when collidine was applied to previously fixed tissues. Paper chromatographic analysis showed that the radioactive compound(s) extracted from tissues by collidine corresponded to noradrenaline and/or closely related compounds. An abstract of this work was sent to the 17th Annual Meeting of the Society for Neuroscience, New Orleans, Nov 16–21, 1987. Tomsig J.L. and Pellegrino de Iraldi A. Abstract 369-11.     

Effect of collidine (2,4,6-trimethylpyridine) on rat pineal gland and vas deferens nerves. Further evidence for a monoamine-releasing effect

In previous work of our laboratory it was demonstrated that collidine (2,4,6-trimethylpyridine) abolishes the core osmiophilia and chromaffin reaction from rat pinal gland and vas deferens nerves. This abolition was apparent when tissues were briefly incubated in collidine or when they wer fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer substance. These and other results strongly suggested that the histochemical effect of collidine was due to depletion of monoamines stored in the vesicles core. To examine this hypothesis we studied in this work the effect of collidine on tissues that have taken up tritiated noradrenaline. It was found that tritium was released very rapidly to the incubation medium when collidine was applied to fresh tissues. This effect was not observed with other commonly used buffers such as cacodylate or phosphate. It was also found that tritium release also occurred, although to a lesser extent, when tissues were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer, and this release was not significant when collidine was applied to previously fixed tissues. Paper chromatographic analysis showed that the radioactive compound(s) extracted from tissues by collidine corresponded to noradrenaline and/or closely related compounds. An abstract of this work was sent to the 17th Annual Meeting of the Society for Neuroscience, New Orleans, Nov 16-21, 1987. Tomsig J.L. and Pellegrino de Iraldi A. Abstract 369-11.     

ZIO staining in synaptic vesicles of the rat pineal nerves after inhibition of serotonin and noradrenaline synthesizing enzymes

Summary Two compartments have been defined in monoaminergic synaptic vesicles: the core or central compartment, storage site for monoamines, and the matrix or outer compartment, of unknown function. The outer compartment reacts with the mixture of zinc iodide-osmium tetroxide (ZIO). This reaction is temperature and time dependent and may be abolished by -SH reagents.The effect of drugs inhibiting the synthesis of serotonin and noradrenaline (stored in the core) on the ZIO reaction in the matrix was studied in synaptic vesicles of rat pineal nerves. The inhibitors of monoamine synthesis abolish or decrease the ZIO reaction directly or in combination with the administration of tyramine. This effect is temperature dependent suggesting that the drugs act on different components of the matrix that react with ZIO at different temperatures. A comparison of the present results with those obtained with -SH reagents seems to indicate that the drugs assayed act, at least in part, by changing the accessibility of-SH groups in vesicle proteins. (An abstract of this paper was presented at the 7th International Congress of Pharmacology, Paris, 1978.)Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, CONICET, and the Secretaría de Ciencia y Técnica, SECYT, ArgentinaWe are grateful to Margarita López de Cáceres for technical assistance, to Adriana Contreras for the electron micrographs and to María Aued de Rau for the illustrations. Rita Cardoni is a fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Effect of K+ and Na+ on calcium-dependent electron-dense particles in the monoaminergic synaptic vesicles of rat pineal nerves fixed in Ca2+-containing solutions

The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+.     

Submicroscopic morphology of granular vesicles in sympathetic nerves of rat pineal body

Summary In the rat pineal body sympathetic nerve endings are characterized by numerous agranular and granular vesicles. The latter mainly include vesicles of about 450 Å diameter, each of which is bounded by a trilaminar membrane of about 75 Å total thickness. The cores of these granular vesicles appear structurally similar to the bounding membranes and are formed by a less dense, globular or vacuolar component around which a more dense component is distributed. The relative distributions of these two structural elements result in a variety of submicroscopic configurations which suggest a morphological continuum that may be related to the cyclical uptake, storage and release of biogenic amines. A tripartite classification of granular vesicles, such as proposed previously, is not supported by these data.This investigation was supported by United States Public Health Service Research Grants NB 05175-01 and AM-0699802. — The capable technical assistance of Mrs. Caroline Wilner is gratefully acknowledged.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Occurrence of uranaffin-positive synaptic vesicles in both adrenergic and non-adrenergic nerves of the rat anococcygeus muscle

Summary The uranaffin reaction in rat anococcygeus muscle, which receives a dual innervation of both adrenergic and non-cholinergic, non-adrenergic nerves was examined. Dense reaction product was observed in the vesicular membranes and/or the cores of some synaptic vesicles in the adrenergic nerve terminals. Occasional vesicles were filled up with dense reaction product. In the prominent population of small clear vesicles, however, no dense reaction product was observed. The number of small granular vesicles in the adrenergic nerve terminals was markedly increased after the administration of 5-hydroxydopamine (5-OHDA). These granular vesicles were moderately stained with uranaffin deposit on the cores but their limiting membranes possessed no uranaffin deposit at all.In the non-adrenergic nerve terminals, on the other hand, uranaffin deposit of variable density was observed on the cores of large granular vesicles but never on their limiting membranes or on the small clear vesicles. There was no change in the axon profiles after the administration of 5-OHDA.The possible occurrence of purines in the cores of large granular vesicles in the non-adrenergic nerves is discussed.     

Ultracytochemistry of the synaptic ribbons in the rat pineal organ

Summary The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO₄-Zn I₂ mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found.In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum.The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets.Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.Grateful acknowledgment is made to Mr. P.-A. Milliquet for technical assistance and to Dr. T. Jalanti (C.M.E., Lausanne) for his help in the use of the X-ray microanalyser.Dedicated to Professor Dr. med. G. Töndury on the occasion of his 70th birthday.     

Interaction of isolated synaptic vesicles with synaptic contacts in the rat brain

The effects of Mg-ATP, EGTA, EDTA and dicyclohexylcarbodiimide on the changes in the intensity of light scattering were studied in rat brain synaptic vesicles (SV) suspended in saccharose-buffer medium. Specific interactions between SV and isolated synaptic junctional complex were observed in the presence of Mg-ATP and calmodulin. An in vitro model of exocytosis is discussed.     

Effect of pyridine and its methylated derivatives on storage by rat pineal nerves of monoaminergic neurotransmitter

In previous work of our laboratory, it was demonstrated that collidine (2-4-6-trimethylpyridine) applied briefly to fresh tissues extracted noradrenaline or closely related compound/s of neuronal origin. This effect gave rise to the abolition of osmiophilia and chromaffin reaction of electron-dense cores of monoaminergic synaptic vesicles and to the extraction of radioactive compounds in tissues that had previously taken up tritiated noradrenaline. In this work, the role of the pyridine ring and its progressive methylation on the monoamine releasing effect was investigated. For this purpose, the effect of pyridine, two picolines (2 and 4 monomethylpyridines) and a lutidine (2-6-dimethylpyridine) was compared to the effect of collidine. It was found that pyridine has a much smaller effect than collidine on the histochemical reactivity of monoaminergic synaptic vesicles and on the extraction of tritiated compounds and that its extent was dependent upon the number of methyl groups incorporated in the pyridine ring.     

Evidence for a gamma-hydroxybutyrate (GHB) uptake by rat brain synaptic vesicles

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of mammalian brain which is derived from GABA. Much evidence favours its role as an endogenous neuromodulator, synthesized, stored and released at particular synapses expressing specific receptors. One key step for GHB involvement in neurotransmission is its uptake by a specific population of synaptic vesicles. We demonstrate that this specific uptake exists in a crude synaptic vesicle pool obtained from rat brain. The kinetic parameters and the pharmacology of this transport are in favour of an active vesicular uptake system for GHB via the vesicular inhibitory amino acid transporter. This result supports the idea that GABA and GHB accumulate together and are coliberated in some GABAergic synapses of the rat brain, where GHB acts as a modulatory factor for the activity of these synapses following stimulation of specific receptors.     

Expression of synaptic vesicle trafficking proteins in the developing rat pineal gland

Adult mammalian pinealocytes contain several synaptic membrane proteins that are probably involved in the regulation of targeting and exocytosis of synaptic-like microvesicles (SLMVs). Immunohistochemical techniques have now demonstrated the spatiotemporal expression pattern of some of these proteins during rat pineal ontogenesis. Various synaptic vesicle trafficking proteins are detectable in proliferating epithelial cells of the pineal anlage even at embryonic day 17.5 (E 17.5), with the exception of syntaxin I (weakly expressed from E 19.5) and dynamin I (whose levels increase markedly during the first postnatal week). Numerous cells exhibiting strong immunoreactivity for synaptobrevin II, SNAP-25, synaptophysin, and munc-18-1 are distributed throughout the increasingly compact gland at E 19.5 and E 20.5; however, their number declines toward the proximal deep part of the organ. Groups of postmitotic cells situated at the surface of the developing gland exhibit marked immunoreactivity for the aforementioned proteins and lie close to the laminin-immunoreactive outer limiting basement membrane or to its remnants in regions of basement membrane dissolution. We also show that synthesis of vimentin and S-antigen seems to begin earlier during pineal development than previously recognized. Thus, synaptic vesicle trafficking proteins are the earliest molecular markers of pinealocyte differentiation known to date, being expressed well before the onset of rhythmic hormone secretion in the pineal gland, where they may play a role in morphogenetic events. Components of the extracellular matrix such as laminin may be critically involved in the upregulation of synaptic membrane protein expression. The dynamin immunostaining pattern indicates that SLMVs of pinealocytes begin to undergo regulated cycles of exo/endocytosis during postnatal week 1.