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.     

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.     

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+.     

The monoaminergic innervation of the rat visual cortex

The intracortical distribution of monoamines, noradrenaline (NA) and serotonin (5-HT), was examined in the visual cortex of the rat with high pressure liquid chromatography (h.p.l.c.) and radioautography. H.p.l.c. measurements showed the densities of both amines to be highest in layer I. The concentration of NA varied considerably in all other layers while the 5-HT concentration decreased with increasing distance from the pial surface. The morphological characteristics of the monoaminergic axon-terminals in the cerebral cortex has been the subject of controversy in recent years. We have used radioautography following topical or intraventricular administration of tritiated amines to examine the ultrastructural features of these terminals in the visual cortex of the rat. Systematic analysis of single sections revealed that more than one-half of the terminals labelled with tritiated NA or 5-HT formed typical synaptic contacts (mostly type I) with dendritic shafts or spines.     

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain.

Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl₂ and 10^?5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.     

Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.     

Electrokinetic properties of synaptic vesicles and synaptosomal membranes.

Using the technique of electrophoretic light scattering, we have measured the electrophoretic mobilities of synaptic vesicles and synaptosomal plasma membranes isolated from guinea-pig cerebral cortex. The electrophoretic mobility of synaptic vesicles is slightly greater than that of synaptosomal plasma membranes. Ca+2 and Mg+2 reduced the mobility of both species to the same extent at physiologically relevant concentrations (0-1 mM) and near-physiologic ionic strength. The extent of the reduction was not large (approximately 6% for synaptic vesicles in the presence of 100 mM KCl) at 1 mM divalent cation concentrations. At concentrations of approximately 2 mM and higher, Ca+2 reduced the mobility of synaptic vesicles more than did Mg/2. A similar but much smaller effect was observed in the case of synaptosomal plasma membranes. The addition of 1 mM Mg+2-ATP had no effect upon synaptic vesicle mobility either in the presence or absence of the ionophores nigericin or valinomycin. These data, together with earlier work (Siegel et al., 1978, Biophys. J. 22:341-346), demonstrate that substantial reduction of the average electrostatic surface charge density is not the most important role of divalent cations in promoting close approach of secretory granules and secretory cell membranes, and that it is certainly not the Ca+2-specific step in exocytosis.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3Hcatecholamine synthesis in discrete brain regions of adult rats

Endogenous noradrenaline levels are elevated in medulla oblongata, mesencephalon, pons and thalamus of adult rats which had been treated with 6-hydroxydopamine on days 1, 2, 8 and 15 after birth. Levels in spinal cord, cerebellum, hippocampus/amygdala and cortex are depressed, whereas no significant changes are observed in striatum, hypothalamus and medulla spinalis. The rate at which medulla oblongata synthesizes tritiated noradrenaline and dopamine from tritiated tyrosine $\text{in}$$\text{vitro}$ is markedly enhanced. No effect was apparent on catecholamine synthesis in hypothalamus. Tritiated noradrenaline synthesis, but not tritiated dopamine synthesis, in the cortex is depressed. These results support the view that neonatal 6-hydroxydopamine treatment causes a degeneration of noradrenaline nerve terminals in the cortex and induces an increase in noradrenaline terminals in the medulla oblongata.     

Monoaminergic Modulation of Spinal Viscero-Sympathetic Function in the Neonatal Mouse Thoracic Spinal Cord

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.     

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     

Synthesis and biological evaluation of pyridine-modified analogues of 3-(2-aminoethoxy)pyridine as novel nicotinic receptor ligands

Analogues of the potent nicotinic receptor agonist 3-(2-aminoethoxy)pyridine substituted at the 5' and 6'-positions of the pyridine ring were synthesized and tested in vitro for nicotinic receptor binding activity (displacement of [(3)H](-)cytisine from whole rat brain synaptic membranes). The substituted analogues exhibited K(i) values ranging from 0.076 to 319 nM compared to a K(i) value of 26 nM for compound 1. Among the compounds tested, 5'-vinyl-6'-chloro substituted 1 was the most potent.     

Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats

Summary Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.     

Effect of carbidine on the content and storage of adrenergic neurotransmitter in the synaptic vesicles]

The influence of carbidine, an original psychotropic drug, on the adrenergic neurotransmitter content and storage in the sympathetic nerves was studied with the use of cytochemical electron microscopy. The influence of carbidine on the uptake of the exogenous noradrenaline (NA) in the synaptic vesicles was also studied. Carbidine was found to be capable to decreasing the NA storage in the synaptic vesicles and failed to block the accumulation of the exogenous NA in the synaptic vesicles.     

Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

Theory of active antidepressants: a nonsynaptic approach to the treatment of depression

Although depression is one of the major neuropsychiatric disorders, the success rate of medication for any drug is about 60%, which means that approximately 40% of the patients does not respond to the initial treatment. The major aim of this review is to provide a possible explanation for the relative inefficacy of currently used antidepressants and to propose a novel mechanism of action, which might improve the success rate of clinical treatment. According to the monoamine theory the most important neurochemical process in depression is the impairment of monoaminergic neurotransmission and the concomitant decrease of extracellular concentration of noradrenaline and/or serotonin. Since the vast majority of monoaminergic varicosities makes no synaptic contact but is able to release transmitters directly into the extrasynaptic space, the monoaminergic neurotransmission is predominantly nonsynaptic in nature. Depression can be regarded, therefore, as a disease, which is developed (at least in part) on the basis of the impairment of nonsynaptic interactions and the effective treatment has to improve this non-conventional communication in the nervous system. The currently used antidepressants (reuptake inhibitors, negative feedback inhibitors, monoamino oxidase inhibitors) can increase the monoamine levels in the extracellular space only if the monoaminergic cells are electrically active and without an action potential-induced vesicular exocytosis these compounds are ineffective. It is proposed that a selective and moderate induction of the carrier-mediated release of NA and 5-HT might be a better therapeutic approach to the treatment of depression, since this new class of antidepressants, the so-called 'active antidepressants' have a mechanism of action, which is independent from the electrical activity of monoaminergic cells, therefore the extrasynaptic concentration of monoamines and thereby the nonsynaptic communication can be enhanced more efficiently.     

Synthesis and evaluation of two 18F-labeled imidazo[1,2-a]pyridine analogues as potential agents for imaging beta-amyloid in Alzheimer's disease

Two new fluorinated imidazo[1,2-a]pyridine derivatives, 6-(2'-fluoroethyl)-2-(4'-dimethylamino)phenylimidazo[1,2-a]pyridine (FEPIP) and 6-(3'-fluoropropyl)-2-(4'-dimethylamino)phenylimidazo[1,2-a]pyridine (FPPIP), were synthesized. The binding affinity for FEPIP and FPPIP to amyloid plaques in human AD cortical tissues was determined. Radiolabeling, in vitro film autoradiography, and micro-PET study were performed with [18F]FPPIP to determine its utility as a radioligand for amyloid plaque imaging in the brain of AD patients.     

Inhibition of neurotransmitter and hormone transport into secretory vesicles by 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine: both compounds act as uncouplers and dissipate the electrochemical gradient of protons.

2-(4-Phenylpiperidino)cyclohexanol (AH-5183) and 2-bromo-alpha-ergocryptine, known inhibitors of the transport of acetylcholine and L-glutamate, respectively, into synaptic vesicles, inhibited the ATP-dependent uptake of dopamine in parallel with the dissipation of the electrochemical gradient of protons in chromaffin granule membrane vesicles. These compounds induced the release of accumulated dopamine from the vesicles. They also inhibited the ATP-dependent formation of the electrochemical gradient of protons in liposomes reconstituted with chromaffin H(+)-ATPase without affecting the activities for ATP hydrolysis, and ATP-dependent uptakes of dopamine, gamma-aminobutyrate, and glutamate into synaptic vesicles. These results indicated that 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine acted as uncouplers in the secretory vesicles.