Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.     

Methodological studies on the uptake of radiocalcium by nerve endings isolated from rat brain.

Different techniques for arresting uptake of ⁴⁵Ca by synaptosomes were compared. 1) Dilution of samples with incubation medium arrested calcium uptake but did not remove extracellularly bound calcium. 2) Dilution with medium containing 0.4 mmol 1^?1 LaCl₃ not only arrested calcium uptake but also prevented calcium efflux and, if enough time was allowed, displaced extracellular calcium. 3) Dilution with medium containing 3 mmol 1^?1 EGTA gave uptake values similar to those obtained with La³⁺, but only if extensive extraction of calcium was prevented by rapid handling of samples. Results obtained after quenching with La³⁺ or EGTA showed that calcium uptake by synaptosomes may be a multiphasic process, which emphasizes the need for techniques that allow for satisfactory time resolution.     

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β₁-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.     

WIN 55,212-2 prevents mechanical allodynia but not alterations in feeding behaviour induced by chronic cisplatin in the rat

Anorexia, nausea/emesis and peripheral sensorial neuropathy are frequent adverse effects associated with chemotherapy. Cannabinoids have been proposed to alleviate these effects, but their preventive properties in long-term experimental models have not been tested. This study was conducted to determine whether or not a cannabinoid agonist (WIN-55,212-2) can prevent anorexia, pica (an indirect marker of nausea in non-vomiting species, consisting of the ingestion of non-nutritive substances such as kaolin) and mechanical allodynia (a marker of peripheral neuropathy) induced by the antineoplastic drug cisplatin chronically administered. Isolated rats with free access to food and kaolin received either saline, cannabinoid vehicle, WIN-55,212-2 (1-2 mg kg(-1)), cisplatin (1-2 mg kg(-1)), or both drugs once per week for five consecutive weeks. Modifications in temperature, body weight gain, food and kaolin intake, and the threshold for mechanical allodynia were recorded. Additionally, the acute psychoactive effects of the cannabinoid (hypomotility, hypothermia, analgesia and catalepsia) were assayed by means of the cannabinoid tetrad. WIN 55,212-2 prevented the development of mechanical allodynia but not anorexia, pica and reduction in weight gain induced by chronic cisplatin. The effect of WIN 55,212-2 was evident even at a dose lacking activity in the cannabinoid tetrad. The preventive effect on cisplatin-induced mechanical allodynia exerted by the cannabinoid could be due to a neuroprotective role, as has been suggested for other conditions. The present results support the interest in the evaluation of cannabinoids for treatment of patients suffering or likely to suffer neuropathic pain.     

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis

1. We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of 45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes). 2. Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 +/- 0.23- and 3.71 +/- 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P less than or equal to 0.1). 3. Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 +/- 5- and 20 +/- 3-fold, respectively, and A23187-induced secretion was increased 1.3 +/- 0.4- and 1.3 +/- 0.1-fold (P less than or equal to 0.01 for both ionophores). 4. The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P less than or equal to 0.01 for sodium greater than or equal to 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1. 5. The rate of 45Ca efflux from neurosecretosomes showed 2.7 +/- 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P less than or equal to 0.01). 6. Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

Effect of parathyroid hormone on the transport of 45Ca2+ and 3H-GABA in nerve endings isolated from the rat cerebral cortex

Parathyroid hormone (PTH) (0.1-10 ng/ml) evokes a dose-dependent increase in 45Ca2+ accumulation in synaptosomes isolated from the rat brain cortex. In the presence of PTH the fast (I sec) potential-dependent 45Ca2+ uptake was less than in the control. PTH had no effect on 3H-GABA uptake by synaptosomes (P2 fraction). Synaptosomes preincubated in the presence of PTH in Ca2+-free medium and transferred into Ca2+-containing normal medium released more 3H-GABA than control synaptosomes. In this case depolarization-evoked 3H-GABA release was diminished.     

Functional characteristics of nerve endings isolated from brain by the Hajos method

A study was made of the functional potentialities of synaptosomes isolated from the brain cortex and lumbar enlargement of the spinal cord. The yield of synaptosomes from the brain cortex amounted to 10 mg (with reference to protein) from 1 g of wet tissue, and that of synaptosomes from the spinal cord was equal to 1/3 of the yield from the brain, with the preparation being strongly contaminated with myelin scraps. Brain synaptosomes were marked by high level of respiration whose magnitude was affected by the agents (ouabain, high concentrations of K+ and benzylpenicillin) that change ion membrane transport. Synaptosomes maintained higher GABA gradient across the plasmatic membrane. Ouabain and potassium depolarization produced a considerable release of GABA and 3H-GABA into the incubation medium. A conclusion is made that the method of Hajos should be rather used for rapid isolation of the synaptosomal fraction from the rat brain cortex.     

Myosin-Va proteolysis by Ca2+/calpain in depolarized nerve endings from rat brain

Myosin-Va is a molecular motor that may participate in synaptic vesicle cycling. Calpain cleaves myosin-Va in vitro at methionine 1141 in the tail domain. We show that intracellular proteolysis of myosin-Va occurs in rat cortical synaptosomes depolarized in the presence of calcium, evidenced by the formation of an 80 k polypeptide that co-migrates in SDS-PAGE with the 80 k fragment produced by the in vitro proteolysis of myosin-Va by calpain. Anti-myosin-Va antibody recognized this polypeptide in Western blots and immunoprecipitated it from synaptosome extracts. Calpastatin, a calpain-specific inhibitor, or leupeptin, a general cysteine protease inhibitor, suppressed or blocked formation of the 80 k polypeptide depending on membrane permeability. We conclude that myosin-Va undergoes intracellular proteolysis by endogenous calpain, when synaptosomes are depolarized in the presence of calcium, at the same cleavage site previously identified in vitro, thus, making it a target for calcium signaling during synaptic activation.     

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.     

Effect of osmolar concentration of culture media on exocytosis in isolated presynaptic nerve endings of rat brain

The effect of hypotonic and hypertonic shock on exocytosis in rat brain synaptosomes was studied using the fluorescent dye acridine orange. It was shown that an increase in medium osmolarity leads to calcium-independent exocytosis. The response of the probe was directly proportional to the amount of osmolithes added. A decrease in medium osmolarity to 230 mOsm led to an increase of acridine orange fluorescence, which is comparable with exocytosis occurring by the action of 15 mM KCl. This effect was independent of calcium concentration. It is assumed that, under hypotonic shock, part of neurotransmitters are released from the vesicular pool.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

The fine structure of nerve endings on rat thyroid follicular cells

Summary Axon profiles in thyroid glands obtained from adult male Wistar rats were studied electron-microscopically, using common and serial thin sections.Bouton profiles of nerve fibers, resembling the terminal or en passant type, often appeared closely associated with vascular smooth muscle cells via basement membranes. These structures are probably adrenergic, since they contained mainly small-core vesicles (mean diameter: 41.2 nm), in addition to a few large-core (mean diameter: 88.4 nm) and flattened vesicles.Nerve fibers containing microtubules and sometimes mitochondria and vesicles were seen lying between basement membranes and follicular cells. The incidence of nerve fiber contacts on profiles of follicular cells was 0.0177±0.0092 (S.D.). Using serial sections, follicles were seen to have up to two nerve endings, separated from the plasma membranes of the follicular cells by a gap of 22 nm. They contained mainly flattened vesicles and several large-core vesicles (mean diameter: 95.1 nm). Small-core vesicles were rarely seen in these nerve endings. Furthermore, subsurface cistern-like rough endoplasmic reticulum was found immediately under the plasma membranes of follicular cells facing membranes of nerve endings. These results suggest that the nerve fibers in contact with follicular cells are different from the adrenergic type.     

Effect of neuroleptics and antidepressants on the process of gamma-aminobutyric acid-H3 uptake by isolated rat brain nerve endings]

The effects of phenothiazine neuropleptics--chlorpromazine, trifluoperazine, fluphenazine and of antidepressants-imipramine and phthoracizine on the GABA-H3 accumulation by synaptosomes of the rat cerebral cortex were studied. All neuroleptics were found to inhibit the process of neurotransmitter uptake by the brain synaptosomes. Antidepressants were less potent. Chlorpromazine had the highest inhibitory effect on GABA uptake and phthoracizine--the lowest. It is suggested that the influence of neurolptics on GABA uptake could play a certain role in the mode of a synaptic action of these drugs.     

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na+, Ca2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na+ (Na(i), as determined with SBFI), and on the rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by an increased permeability of presynaptic Na+ channels, with veratridine, or by an increased permeability of presynaptic Ca2+ channels with high K+, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC(50)'s for carbamazepine and vinpocetine to inhibit veratridine-induced [3H]Glu release are 200 and 2 microM, respectively. Consistently 150 microM carbamazepine and 1.5 microM vinpocetine reduce the veratridine-induced rise in Na(i) in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na+ channel mediated responses, namely the rise in Na(i) and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca2+ via presynaptic Ca2+ channels, such as the release of [3H]Glu and the rise in Ca(i) induced by high K+, are insensitive to 300 microM carbamazepine and slightly reduced by 5 microM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.