Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

[Membrane cholesterol oxidation effects on synaptic vesicle cycle in frog (RANA ridibunda) motor nerve terminals].

In experiments on frog (Rana ridibunda) neuromuscular junction the influence of cholesterol oxidation on the presynaptic vesicular cycle was investigated. Application of cholesterol oxidase (1 u. a.) during 1/2 hour led to the oxidation of - 0.007 mg cholesterol per 1 g tissue and reduced stability of lipid rafts in the nerve terminals. Using electrophysiological techniques it was shown that the cholesterol oxidation decreases the evoked neurotransmitter release. In experiments with fluorescent FM-dyes the depression of the synaptic vesicles exo-endocytosis and the dispersion of synaptic vesicles clusters were revealed. Comparative analysis of electrophysiological and optical data, as well as experiments with water soluble quencher of FM-dye indicated the possibility of some neurotransmitter release by "kiss-and-run" pathway, when short-lived fusion pore is formed. It was concluded that cholesterol oxidation inhibit evoked exocytosis, and also synaptic vesicle delivery from reserve pool to cites of exocytosis probably by break of the clusterization. Perhaps the synaptic vesicles of recycling pool release the neurotransmitter using the kiss-and-run mechanism.

Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-ß-cyclodextrin: the role of vesicular acetylcholine transporter.

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-ß-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

The role of cholesterol in the exo- and endocytosis of synaptic vesicles in frog motor nerve endings.

Experiments on frog neuromuscular preparations using electrophysiological (two-electrode voltage clamping) and optical (with the fluorescent endocytic stain FM1-43) methods were performed to study the importance of membrane cholesterol in the exo- and endocytic cycle of synaptic vesicles (SV) in motor nerve endings in conditions of prolonged rhythmic stimulation of the motor nerve (20 impulses/sec, 3 min). Extraction of cholesterol from the superficial plasma membranes using methyl-beta-cyclodextrin (1 mM) led to marked changes in SV recycling. There was weakening of SV exocytosis and suppression of processes leading to the recovery of SV populations with rapid readiness to release neurotransmitter. When cholesterol was leached from the outer membranes and the membranes of SV undergoing recycling, these effects were supplemented by impairments to SV endocytosis and recycling. Thus, plasma membrane cholesterol plays a key role in the processes of exocytosis, while the efficiency of endocytosis depends on cholesterol in SV membranes.

[The role of cholesterol in exo- and endocytosis of the synaptic vesicles at the frog motor nerve terminal].

In experiments on the frog neuro-muscular preparations using electrophysiological (two electrode fixing of potential) and optical (fluorescent endocytic dye FM1-43) methods, the value of surface cholestertol for exo-endocytic cycle of synaptic vesicles at the prolonged rhythmic activity (20 Hz--3 minutes) was investigated. It is shown that extraction of cholesterol from surface membranes by methyl-betta-cyclodextrin (1 mM MCD) leads to the expressed shifts in recycling of synaptic vesicles. Exocytosis of vesicles is decreased, and oppression of processes leading to restoration of the number of vesicles of ready releasable pool is observed. Cholesterol replacement from external membranes and membranes of recycling synaptic vesicles in addition to above described effects breaks processes of endocytosis and recycle of synaptic vesicles. Thus, in the processes of exocytosis, the key role is played by cholesterol of plasmatic membranes, and endocytosis critically depends on the amount of cholesterol in the membranes of synaptic vesicles.

SNAP25 is a pre-synaptic target for the depressant action of reactive oxygen species on transmitter release.

Reactive oxygen species (ROS) participate in various physiological and pathological processes in the nervous system, but the specific pathways that mediate ROS signalling remain largely unknown. Using electrophysiological techniques and biochemical evaluation of isolated fusion proteins, we explored the sensitivity to standard oxidative stress of the entire synapse, the pre-synaptic machinery and essential fusion proteins underlying transmitter exocytosis. Oxidative stress induced by H(2)O(2) plus Fe(2+) inhibited both evoked and spontaneous quantal release from frog or mouse motor nerve endings, while it left post-synaptic sensitivity unchanged. The depressant effect of H(2)O(2) on acetylcholine release was pertussis toxin-insensitive, ruling out G-protein cascades. Experiments with ionomycin, a Ca(2+) ionophore, revealed that ROS directly impaired the function of releasing machinery. In line with this, SNAP25, one of three essential fusion proteins, showed a selectively high sensitivity to the oxidative signals. Several ROS scavengers enhanced evoked synaptic transmission, consistent with tonic inhibition by endogenous ROS. Our data suggest that ROS-induced impairment of releasing machinery is mediated by SNAP25, which appears to be a pre-synaptic ROS sensor. This mechanism of ROS signalling could have widespread implications in the nervous system and might contribute to the pathogenesis of neurodegenerative diseases.

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction.

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2',7'-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction.

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two-electrode voltage-clamp technique, at frog neuromuscular junctions, the action H2O2 on end-plate currents (EPC) was studied to determine the targets for this membrane-permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 microM) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high-frequency stimulation. Pro-oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N-acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4beta-phorbol 12-myristate, 13-acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

[Effect of hydrocortisone on purine actions in the nerve-muscle preparation]. / Vliianie gidrokortizona na moduliruiushchie éffekty purinov v nervno-myshechnom soedinenii.

Daily administration of hydrocortisone first increased and then decreased amplitude of multiquantal end-plate currents induced by motor nerve stimulation. The initial facilitating phase of the hormone action was accompanied by elimination of the ATP pre-synaptic effect. Later the inhibitory effect of the ATP becomes restored. The counteraction of the ATP effect was reproduced in the isolated muscle bathed in the saline with hydrocortisone which suggests a non-genomic action of the hormone on pre-synaptic P2 receptor. The data obtained suggest that prevention of the ATP inhibitory action might be a component of a facilitating acute stress reaction.