Compartmentalization of monoaminergic synaptic vesicles in the storage and release of neurotransmitter.

Monoaminergic nerves are characterized by the presence of a population of small synaptic vesicles (40-60 nm in diameter) containing a few large vesicles (80-90 nm in diameter). Thus, although both types of vesicles contain monoamines, the small vesicles must be considered as the organoid responsible for the storage and release of the neurotransmitter, whereas the large ones possibly are involved in the modulation of the process. The small vesicles are electron-lucent or have an osmiophilic electron-dense core that is always linked to the vesicle membrane. Considering morphological and histochemical evidence under different experimental conditions, we proposed the existence of two compartments in the small vesicles: the core and the matrix, corresponding respectively to the electron-dense core and the electron-lucent space between the core and the vesicle membrane in osmium tetroxide fixations. The sizes of both compartments are inversely related, i.e., the smaller the core, the larger the matrix and vice versa. The core even disappears, giving way to a small electron-lucent vesicle made exclusively by the matrix. Thus, the matrix is a constant component of the vesicle, whereas the core is a transient one. Each compartment has a different pool of amine: a loosely bound, easily releasable pool in the matrix and a tightly bound, more resistant pool in the core. These two pools subserve, respectively, a tonic or phasic release of the neurotransmitter, correlated with a tonic or phasic stimulation of the receptor. The core may be considered as a storage or reserve pool. Experimental evidence from our laboratory supports the concept that different mechanisms are operative in both compartments in the release of the neurotransmitter. For instance, a Ca2(+)-independent release would be primarily concerned with the neurotransmitter contained in the matrix, and a Ca2(+)-dependent efflux would be primarily related with the neurotransmitter stored in the core. However, it still must be established that a simple relationship exists between each kind of stimulus and each vesicle compartment, rather than both compartments being integrated in a dynamic functional unit.

Possible involvement of Na+,K(+)-ATPase inhibition in neurotransmitter release induced by collidine.

Previous histochemical studies have demonstrated that collidine abolished the osmiophilia and the chromaffin reaction of the synaptic vesicles, an effect which was ascribed to the release of stored neurotransmitters. Other studies indicated a relationship between the inhibition of Na+,K(+)-ATPase and the release of neurotransmitters. In the present study it is shown that collidine inhibits cation-stimulated ATPase activities of the brain synaptosomal membranes. This favours the idea that neurotransmitter release induced by collidine could be due to the inhibition of Na+,K(+)-ATPase activity.

Selective axonal argentaffin staining in rat central nervous system after protein mercuration.

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.

Differential staining of two subpopulations of Purkinje neurons in rat cerebellum with acid dyes.

We present a new method that stains differently two subpopulations of Purkinje cells in the adult rat. Deparaffinized sections of cerebella, fixed by perfusion with buffered glutaraldehyde or Bouin's fluid were stained with 0.5% light green in 50% ethanol (10-30 min). The excess dye was removed with saturated aqueous picric acid (10-30 min). At this point some Purkinje cells appeared as lightly stained neurons, while others were strongly stained. Slides were immersed in 0.5% aqueous acid fuchsin for approximately 1 min until the lightly stained neurons acquired a red color. Following immersion in 1% phosphotungstic acid, slides were rapidly dehydrated in ethanol, passed to xylene and mounted in Canada balsam. Two subpopulations of Purkinje cells differing in their protein content in somata and proximal dendrites stained differentially by this method. They occurred in all coronal and sagittal sections and in patches or stripes. Their relative proportion varied from lobule to lobule. A second staining method used potassium permanganate as the sole staining reagent. The staining reagent can be used on sections previously stained with the acid dyes. Purkinje cells appeared as subsets of brownish to deep brown stained neurons, the latter ones corresponding to green stained cells in the dichromic method. The results obtained indicated that the subpopulations reflect real differences among individual neurons and are not artifacts. The technique holds promise for identifying and localizing sub-sets of Purkinje cells differing in their protein content under normal and experimental conditions and for their further characterization by combined staining and histochemical procedures.

Effect of dyskinetoplastic agents on ultrastructure and oxidative phosphorylation in Crithidia fasciculata.

Ethidium bromide (EB) is an intercalating agent which binds specifically to the kinetoplast (mitochondrial) DNA (kDNA) of trypanosomatids. Accordingly, EB inhibits DNA replication, thus inducing dyskinetoplasty. Since in eukariotic organisms mitochondrial DNA encodes the genetic information for cytochromes b, aa3 and F0F1 ATPase, it seemed of interest to establish whether a similar effect occurs in Crithidia fasciculata, a trypanosomatid used for assay of potential trypanocidal drugs. Culturing of C. fasciculata in the presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic arc shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskinetoplasty decreased crithidia respiration and oxidative phosphorylation, as indicated by the lower ATP level, ATP/ADP ratio and adenylate energy charge. The interference of EB with kinetoplastic constituents synthesis was confirmed by the lack of action of EB on crithidia in the stationary phase of growth, that ruled out direct inhibition of oxidative phosphorylation enzymes. The lipophilic o-naphthoquinone beta-lapachone produced structural alterations in kinetoplast membranes, that correlated with inhibition of oxidative phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries.

The heart has an endocrine activity which depends on the secretion of a natriuretic, diuretic and hypotensive factor contained in osmophilic, secretory granules localized in the myocardiocytes and called "atrial specific granules" (the atrial natriuretic factor, ANF). In this paper, the relationship between these specific granules and renovascular hypertension elicited by the constriction of both renal arteries was investigated at the electron microscope level during the acute, subacute and chronic phases of hypertension. Male Wistar CHbb THOM rats were divided in three groups: 1) clipped rats; 2) sham operated rats; 3) ether anesthesia as unique manoeuver 48 h before decapitation. Blood pressure increased progressively after the constriction of both renal arteries. The atrial specific granules were not affected by ether anesthesia alone; 48-72 h after clipping the granules almost disappeared and this situation persisted up to the 6th week. In sham operated rats the picture was very similar to the clip rats 48 and 72 h after surgery (severe granule disappearance); in contrast, at one, two and six weeks after surgery, the granularity of cardiomyocytes in sham rats was absolutely restored. It is concluded that: 1) similarities in morphology of atrial specific granules in sham and clip rats 48 and 72 h after surgery would suggest that stress plays a primary role in determining the observed images; 2) thereafter, the contrast between sham and clip rats 1, 2 and 6 weeks after surgery would indicate that the ANF is linked to the subacute and chronic regulation of renovascular hypertension.

Atrial natriuretic factor in two kidney--two clip renovascular hypertension in the rat.

Hig levels of circulating atrial natriuretic factor (ANF) have been reported in several physiopathologic conditions like hypertension, heart and renal failure, pregnancy and high sodium intake. Nevertheless, neither relationships with water-sodium space regulation nor the role of an ANF vascular relaxant effect have been yet defined. The aim of present experiments was to characterize the contribution of circulating ANF and its vascular relaxing effects in the two kidney-two clip (2K2C) experimental model of renovascular hypertension. Complementary, plasma metabolites nitrite/nitrate of nitric oxide (NO) was examined because of mediation for both (NO an ANF) through cGMP. Three results showed (two-four weeks after surgery): indirect systolic blood pressure (mmHg), 186 +/- 4 in HT and 122 +/- 1 in SH (p < 0.001); a significant increase of plasma ANF (fmol/ml) in HT (n = 7, 1221 +/- 253) vs. SH (n = 9, 476 +/- 82; p < 0.02). Nitrate/nitrite plasma concentrations (mumol/l) were mpt different between SH and. The relaxant effect of ANF (10(-9), 10(-8) and 10(-7) M) on phenylephrine (3,5 x 10(-6) M) contracted rings from HT rats was smaller than SH rats (10(-8) M, p < 0.05). Contractions to phorbol 12, 13-dibutyrate (seven weeks after surgery) were significantly higher in rings from HT rats (p < 0.001). We conclude: 1) in addition to decreased granularity in atrial myocardiocytes, high circulating values of ANF here described suggest an increased turnover of the peptide in 2K2C hypertensive rats; 2) lower significant vascular relaxant effects in HT rats would indicate down regulation of ANF receptors in this model; the latter would derive from high plasma ANF concentration and, tentatively, because of greater activity of protein kinase C in the vascular wall; 39 similar values of plasma nitrite/nitrate in SH and HT rats would indicate a comparable NO circulating availability in both groups.

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

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.)

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.

A silver-reducing component in rat striated muscle. I. Selective localization at the level of the terminal cistern/transverse tubule system. Light and electron microscope studies with a new histochemical procedure.

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (Hg--Ag) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 degrees C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

Mg2+/Ca(2+)-ATPase activity is not enriched in synaptic vesicles isolated from rat cerebral cortex.

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg2+/Ca(2+)-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg2+/Ca(2+)-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg2+/Ca(2+)-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg2+ and Ca(2+)-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg2+/Ca(2+)-ATPase is a good marker for synaptic vesicles.

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.

Retinal alterations induced by continuous light in immature rats. II. Zinc iodide-osmium tetroxide (ZIO) reactive sites in the rod outer segments.

Immature albino rats were subjected to (a) continuous illumination for 5-9 days, or (b) continuous illumination followed by prolonged darkness. Their electroretinographic responses and the ultrastructural characteristics of the rod outer segments, as revealed by a mixture of zinc iodine-osmium tetroxide (ZIO) at different temperatures, were studied and compared with those of a control group maintained in a cyclic rhythm of light and darkness. Noteworthy differences in the distribution of ZIO reactive sites were observed in the rats exposed for 5-9 days to continuous illumination (no electroretinographic responses) as compared with normal controls. At 4 degrees C, ZIO staining was negative in the rods of illuminated rats whereas at 20 and 60 degrees C it was positive inside the tubular and vesicular structures. After prolonged darkness, in rats with a partial electroretinographic recovery and ultrastructural restoration, the ZIO reaction showed a similar pattern to that observed in the control group, ZIO deposits being found both in the intra- and extradiscal spaces.

Two pools of amines in synaptic vesicles of rat pineal nerves.

Two pools of amines (noradrenaline and serotonin) are characterized by histochemistry and electrical stimulation in the synaptic vesicles from rat pineal nerves : a loosely bound pool located in the matrix and a tightly bound pool located in the core. A similar distribution is adopted by the false transmitter formed by the administration of 5-hydroxydopamine. The behaviour of both pools under electrical stimulation and in resting conditions is compatible with the idea that the amines stored in the core form a reserve pool and the amines stored in the matrix correspond to a functional pool, which can be released spontaneously and by electrical stimulation, being replaced by the transmitter newly synthesized or by the amines stored in the core. (A presentation of these results was made at the III International Symposium on Nervous Transmission, Helsinki, Finland, 1979).